Structures of NF-κB p52 homodimer-DNA complexes rationalize binding mechanisms and transcription activation

  1. Wenfei Pan
  2. Vladimir A Meshcheryakov
  3. Tianjie Li
  4. Yi Wang
  5. Gourisankar Ghosh
  6. Vivien Ya-Fan Wang  Is a corresponding author
  1. Faculty of Health Sciences, University of Macau, China
  2. Department of Physics, Chinese University of Hong Kong, Hong Kong
  3. Department of Chemistry and Biochemistry, University of California, San Diego, United States
  4. MoE Frontiers Science Center for Precision Oncology, University of Macau, Macao
  5. Cancer Centre, Faculty of Health Sciences, University of Macau, China

Abstract

The mammalian NF-κB p52:p52 homodimer together with its cofactor Bcl3 activates transcription of κB sites with a central G/C base pair (bp), while it is inactive toward κB sites with a central A/T bp. To understand the molecular basis for this unique property of p52, we have determined the crystal structures of recombinant human p52 protein in complex with a P-selectin(PSel)-κB DNA (5′-GGGGTGACCCC-3′) (central bp is underlined) and variants changing the central bp to A/T or swapping the flanking bp. The structures reveal a nearly two-fold widened minor groove in the central region of the DNA as compared to all other currently available NF-κB-DNA complex structures, which have a central A/T bp. Microsecond molecular dynamics (MD) simulations of free DNAs and p52 bound complexes reveal that free DNAs exhibit distinct preferred conformations, and p52:p52 homodimer induces the least amount of DNA conformational changes when bound to the more transcriptionally active natural G/C-centric PSel-κB, but adopts closed conformation when bound to the mutant A/T and swap DNAs due to their narrowed minor grooves. Our binding assays further demonstrate that the fast kinetics favored by entropy is correlated with higher transcriptional activity. Overall, our studies have revealed a novel conformation for κB DNA in complex with NF-κB and pinpoint the importance of binding kinetics, dictated by DNA conformational and dynamic states, in controlling transcriptional activation for NF-κB.

Editor's evaluation

This manuscript provides an important structural and biophysical characterization of several complexes of the p52 homodimer of NF kB and different DNA binding sites. The main topic is the investigation of why the central base pair(s) have a strong influence on the transcriptional activity of the homodimer. The authors correlate structural changes with measurements of kinetic on and off rates to develop a model that explains the differences in activity and supports their interpretation with MD simulations, however, some technical issues regarding differences in the ITC and BLI measurements remain unresolved. The paper will be of interest to scientists working on understanding transcriptional regulation.

https://doi.org/10.7554/eLife.86258.sa0

Introduction

The binding of transcription factors (TFs) to their specific DNA response elements in the promoters/enhancers of target genes is the key event regulating gene transcription and consequent cellular processes. For proper gene expression, TFs must interact selectively at the correct place and time and assemble into high-order complexes with specific DNA sequences and cofactors (Natoli et al., 2005; Mulero et al., 2019). In eukaryotic genomes, the ability of TFs to select a small subset of relevant binding sites out of the large excess of potential binding sites within the genomes is the foundation upon which transcriptional regulation is built. Structural studies have provided valuable information on how various DNA binding domains recognize their cognate DNA binding sites at atomic resolution (Garvie and Wolberger, 2001). However, how TFs discriminate between closely related, but biologically distinct, DNA sequences are not well understood.

The nuclear factor κB (NF-κB) family of TFs regulates diverse biological responses (Zhang et al., 2017). Mammalian NF-κB is assembled combinatorially from five subunits, p50/NF-κB1, p52/NF-κB2, RelA/p65, c-Rel, and RelB, into homo- and heterodimers which bind to specific DNA sequences, known as κB site or κB DNA. All five subunits share a highly conserved region at their N-termini, referred to as the Rel homology region (RHR), and the three-dimensional structures of the RHR are also highly conserved among these proteins. The RHR is roughly 300 residues in length and contains the N-terminal domain (NTD), dimerization domain (DD), and nuclear localization signal (NLS). The DD alone mediates protein homo- and heterodimerization; the NTD and DD together are responsible for DNA binding; and the NLS region is flexible in solution and together with the DD forms the binding sites for the inhibitor of NF-κB (IκB) proteins.

The NF-κB proteins can be further divided into two subclasses: the p50 and p52 subunits belong to class I by virtue of their lack of a transcriptional activation domain (TAD). The other three subunits, RelA, c-Rel, and RelB, constitute class II with every member containing a TAD at its C-terminus. Mature p50 and p52 subunits are generated via incomplete proteolysis of their precursor proteins p105 and p100 (Figure 1—figure supplement 1A), respectively. Therefore, p50 and p52 possess a short glycine-rich region (GRR) at their C-termini.

The initial discovery and characterization of several physiological κB DNAs established the pseudo-symmetric consensus sequence as 5′-G−5G−4G−3R−2N−1W0Y+1Y+2C+3C+4-3′ (Lenardo and Baltimore, 1989), where R=purines, N=any nucleotides, W=either A or T, and Y=pyrimidines. The subsequent identification of new NF-κB-DNA binding sites broadened the consensus to 5′-G−5G−4G−3N−2N−1N0N+1N+2C+3C+4-3′ (Chen and Ghosh, 1999; Mulero et al., 2019). The critical features of the consensus κB DNA sequence are the presence of a series of G and C bases at the 5′ and 3′ ends, respectively, while the bases at the central region can vary. X-ray structures of various NF-κB dimers in complex with different κB DNAs revealed conserved protein-DNA recognition modes for κB DNA that follows the consensus sequence (Müller et al., 1995; Ghosh et al., 1995; Cramer et al., 1997; Chen et al., 1998b; Huang et al., 2001; Moorthy et al., 2007; Fusco et al., 2009; Chen et al., 1998a; Chen et al., 2000; Escalante et al., 2002; Berkowitz et al., 2002; Chen-Park et al., 2002; Panne et al., 2007). The RHR of each monomer binds to half of a κB DNA, called half-site. A set of conserved amino acid (aa) residues mediate base-specific contacts to the 5′ and 3′ flanking G and C bases; the inner, more variable bases participate in important, but less base-specific interactions. The length of κB DNAs varies from 9 to 11 base pairs (bps). The p50 and p52 subunits bind to a 5 bp half-site (5′-GGGNN-3′), while the RelA and c-Rel subunits prefer a 4 bp half-site (5′-NNCC-3′). Following this binding pattern, both the p50:p50 and p52:p52 homodimers prefer an 11 bp κB site comprising of two 5 bp half-sites separated by a central bp (5+1+5 bps), such as the IL-6-κB site (5′-GGGATTTTCCC-3′). On the other hand, RelA:RelA and c-Rel:c-Rel homodimers bind 9 bp κB sites containing two 4 bp half-sites (4+1+4 bps), such as the IL-8-κB site (5′-GGAATTTCC-3′). Heterodimers containing one p50 or p52 subunit, such as p50:RelA and p52:RelB, recognize a 10 bp κB site (5+1+4 bps), that is, the HIV-κB site (5′-GGGACTTTCC-3′) and IFN-β-κB site (5′-GGGAAATTCC-3′), in which the central bp lies at the pseudo-dyad axis of the dimer and is not directly contacted by the protein.

Genome-wide NF-κB-DNA motif identification studies revealed that NF-κB associates not only with consensus κB DNAs, but also with sequences containing only one half-site consensus, and even some sequences with no consensus (Lim et al., 2007; Martone et al., 2003; Zhao et al., 2014). In vitro binding experiments have been carried out to classify κB DNAs according to their binding specificity for different NF-κB dimers. The binding affinity displayed by various NF-κB dimers for distinct κB DNAs does not necessarily correlate with what occurs during regulation of gene expression in vivo. For example, the p50:RelA heterodimer binds tightly to most κB DNAs, whereas RelA:RelA and c-Rel:c-Rel homodimers bind many of the same sequences with relatively low affinity. However, detailed genetic experiments have shown that some genes are activated only in the presence of one or a subset of NF-κB subunits, such as mice lacking c-Rel exhibit defects in IL-2 and IL-12 expression (Köntgen et al., 1995; Hoffmann et al., 2003). In addition to specific gene activation, NF-κB dimers are also known to repress transcription. The RelA and p50 dimers have been shown to repress the expressions of nrp1 gene, which is essential for osteoclast differentiation, and the interferon-stimulated response element, respectively (Cheng et al., 2011; Hayashi et al., 2012). Both of these sites also display only half-site similarity to the κB DNA consensus. Structural and biochemical analyses of NF-κB-DNA binding have also revealed the existence of a large number of κB DNAs that display relatively similar affinities compared with κB consensus even though they lack one consensus half-site entirely (Ghosh et al., 2012; Siggers et al., 2011). Therefore, in vitro data do not fully capture the complexity of DNA recognition and gene regulation by NF-κB in cells.

NF-κB p52 is generated from the precursor protein p100 (Figure 1—figure supplement 1A), a tightly regulated process that requires specific stimuli. Unregulated p100 processing into p52 results in multiple myeloma and other lymphoid malignancies, which is detrimental to normal cellular function (Courtois and Gilmore, 2006; Annunziata et al., 2007; Keats et al., 2007). We previously demonstrated that the p52:p52 homodimer could sense a single bp change from G/C to A/T at the central position of a κB DNA (Wang et al., 2012). The p52:p52 homodimer binds both κB DNAs; but only in the case of the G/C-centric DNA, p52:p52 homodimer can associate with its specific cofactor Bcl3 (p52:p52:Bcl3 complex) and activate transcription by recruiting histone acetyltransferases. When bound to the A/T-centric DNA, the same p52:p52:Bcl3 complex represses gene transcription through the recruitment of histone deacetylases. It is intriguing that the identity of a non-contacted nucleotide should have such a drastic effect on transcriptional selectivity. Leung et al. reported that the transcriptional activity of the RelA:RelA homodimer upon binding to the IP-10-κB DNA (5′-GGGAAATTCC-3′) and the MCP-1-κB DNA (5′-GGGAATTTCC-3′) are different; a single bp difference between the A-centric IP-10-κB and T-centric MCP-1-κB DNAs alters the genes’ responsiveness to RelA (Leung et al., 2004). Taken together, these reports strongly suggest that NF-κB transcriptional outcomes are coded within specific κB DNA sequences. Even small changes in the promoter-specific κB DNAs, which do not alter the overall NF-κB binding affinity, might alter the gene expression profiles. Although structural studies have revealed a stereochemical mechanism of how NF-κB dimers bind κB DNAs, the effect of DNA conformation on complex formation remains underappreciated and it requires solid understanding of both structure and dynamics of κB DNAs to elucidate such a mechanism.

In the present study, we determined the crystal structures of the p52:p52 homodimer in complex with the natural G/C-centric PSel-κB DNA (5′-GGGGTGACCCC-3′) and two related DNAs where the central three positions were varied, named as PSel (mutant A/T-centric) (5′-GGGGTAACCCC-3′) and PSel (−1/+1 swap) (5′-GGGGAGTCCCC-3′). PSel is a known target gene regulated by the p52:p52:Bcl3 complex in cells (Pan and McEver, 1995; Wang et al., 2012); however, the PSel (mutant A/T-centric) and (−1/+1 swap) κB DNAs reduced the transcriptional activity significantly. All three structures revealed a widening of the DNA minor groove in the central region compared to all previously known structures of NF-κB-(A/T-centric)-DNA complexes. MD simulations showed free DNAs exist in distinct preferred conformations, and the p52:p52 homodimer induces the least amount of conformational changes on the more transcriptionally active Psel (natural G/C-centric) κB DNA which has an intrinsically widened minor groove. In vitro experiments further demonstrated that the binding kinetics, rather than the binding affinity, is correlated with their transcriptional activities. The combination of structural, MD simulations, and biochemical studies presented here provides new insights into allosteric control by closely related κB DNAs on NF-κB-dependent transcriptional specificity.

Results

The central base pairs in PSel-κB DNA regulate p52:p52:Bcl3 transcriptional activity

Structures of several NF-κB dimers in complex with various κB DNAs have been reported over the past 25 years. In all these structures, the DNA sequences contain A/T-centric κB sites (Figure 1—figure supplement 2E; Ghosh et al., 1995; Müller et al., 1995; Cramer et al., 1997; Chen et al., 1998a; Huang et al., 2001; Moorthy et al., 2007; Fusco et al., 2009; Chen et al., 1998b). The PSel-κB DNA (5′-GGGGTGACCCC-3′) (the central bp is in red color, bps at ±1 positions are underlined), a natural binding site known to be specifically regulated by the p52:p52:Bcl3 complex (Pan and McEver, 1995; Wang et al., 2012), is distinctive from the canonical κB sites not only at the central position but also the two flanking positions. Whereas p50 and other subunits prefer an A:T at −1 and T:A at +1 positions, such as the MHC-κB site (5′-GGGGATTCCCC-3′), PSel-κB contains T:A and A:T at the equivalent positions, respectively. We mutated the central and flanking bps to generate PSel (mutant A/T-centric) (5′-GGGGTAACCCC-3′) and (−1/+1 swap) (5′-GGGGAGTCCCC-3′) DNAs. Transcriptional activity of the p52:p52:Bcl3 complex was measured for these three and MHC-κB sites using a luciferase reporter-based assay. The natural PSel luciferase reporter could be activated by endogenous NF-κB with co-expression of Bcl3 followed by lipopolysaccharide stimulation (Figure 1A). To investigate the effects of PSel (mutant A/T-centric) and (−1/+1 swap) on transcriptional activity, luciferase reporter constructs with the variants or MHC-κB site were co-transfected with p52 and Bcl3 expression plasmids. PSel (mutant A/T-centric) showed twofold reduced reporter activity, while both PSel (−1/+1 swap) and MHC-κB showed drastically reduced transcriptional activity as compared to the natural PSel-κB (Figure 1B; Figure 1—figure supplement 1C). These results suggest that the bp identity at all three positions in the central region is critical in determining transcriptional activity of the p52:p52:Bcl3 complex, which is in line with our previous study that the central bp of κB DNAs plays critical roles in transcriptional regulation (Wang et al., 2012).

Figure 1 with 2 supplements see all
Crystal structures of p52:p52 homodimer in complex with PSel-κB DNA variants reveal distinct signatures.

(A) The natural G/C-centric PSel luciferase reporter was activated by endogenous NF-κB with LPS stimulation and Bcl3 co-expression. The data were analyzed from three independent experiments performed in triplicate. RLU, relative luciferase unit. *p<0.05; **p<0.01 (t test). Error bars represent standard deviation (SD). (B) Luciferase reporter activity driven by co-expression of p52 and Bcl3 was reduced when the natural G/C-centric PSel site was mutated to A/T-centric or −1/+1 swap sites; and the MHC luciferase reporter was not activated by p52:p52:Bcl3 complex. The data were analyzed from three independent experiments performed in triplicate. *p<0.05; **p<0.01; ***p<0.001; n.s., not significant (t test). Error bars represent SD. (C) Overall structure of p52:p52 in complex with the natural G/C-centric PSel-κB DNA. (Left) Ribbon diagram showing the entire complex viewed down the DNA helical axis. The two p52 monomers are shown in orange (monomer I) and green (monomer II), respectively; and the DNA duplex is shown in blue; (Right) View of the complex after rotating 90° along the vertical axis. (D) Overlay p52:p52 homodimers in three PSel-κB DNA variants by their dimerization domain (DD). Monomer I is shown in tv_orange, bright orange, and light orange; monomer II is shown in forest, tv_green, and lime in the natural G/C-centric, mutant A/T-centric, and −1/+1 swap complexes. All three structures are presented as backbone traces. (E–G) Structure of the 18 bp PSel-κB DNAs with (E) natural G/C-centric (blue), (F) mutant A/T-centric (light pink), and (G) −1/+1 swap (ruby). The DNA bps as observed in the co-crystal structures are shown in filled sticks. The view is onto the central minor groove. The nucleotide sequences used in co-crystallization are shown at the bottom, with κB DNA underlined and numbering scheme indicated above; the central position 0 is highlighted in red, and the swap of −1 and +1 positions is highlighted in green. (H) Overlay of natural G/C-centric, mutant A/T-centric, and−1/+1 swap PSel-κB DNAs in (E–G). (I) Table showing minor groove widths and major groove depths (Å); the ideal B-form DNA was built using Coot program (Emsley and Cowtan, 2004; Emsley et al., 2010) based on the sequence of PSel-κB DNA. The minor groove widths at the central region from position −1 to +1 are shown in red, and the corresponding major groove depths are shown in green. Geometrical parameters and the helical axes were calculated with Curves+. Groove widths are measured as minimal distances between the backbone spline curves passing through the phosphorus atoms; values are given at base pair levels and halfway between these levels (Blanchet et al., 2011; Lavery et al., 2009). LPS, lipopolysaccharide.

Widened minor groove in PSel-κB DNA in complex with NF-κB p52:p52 homodimer

Since only p52 mediates DNA interactions in the p52:p52:Bcl3 complex (Figure 1—figure supplement 1D; Bours et al., 1993), we focused our study on (p52:p52)-DNA and speculated that the observed transcriptional differences could be due to different structural features of (p52:p52)-DNA complexes. We solved the crystal structures of p52:p52 homodimer in complex with all three PSel-κB DNAs (Figure 1C–H; Table 1). The p52 protein works as a bridging factor between target DNAs and Bcl3; therefore, a recombinant p52 protein (aa 1–398) which could form complex with Bcl3 was co-crystallized with the DNAs (Figure 1—figure supplement 1E–H). This p52 construct contains most of the GRR region which was not included in any previous NF-κB structures (Figure 1—figure supplement 1B, Figure 1—figure supplement 2E); however, no electron density was observed for the C-terminal part (aa 330–398) in the structures. The length of PSel-κB DNAs used in the co-crystallization trials ranged from 12 to 20 bp (Supplementary file 1). p52 (aa 1–398) protein co-crystallized with 18 and 20 bp DNAs but diffracted to a higher resolution with the 18 bp DNA.

Table 1
Summary of crystallographic information.
Structurep52-PSel(natural G/C-centric)(7CLI)p52-PSel(mutant A/T-centric)(7VUQ)p52-PSel(−1/+1 swap)(7VUP)p52-PSel(mutant 13-mer A/T-centric)(7W7L)
p52 constructaa 1–398aa 1–398aa 1–398aa 1–327
DNA length18 bp18 bp18 bp13 bp
Data collection
Wavelength (Å)0.978520.9791830.9791830.979183
Resolution range (Å)45.63–3.00 (3.16–3.00)46.48–3.10 (3.27–3.10)46.85–3.40 (3.58–3.40)48.77–3.00 (3.16–3.00)
Space groupP212121P212121P212121P6222
a, b, c (Å)84.50, 85.37, 140.2984.45, 84.63, 139.4383.99, 84.29, 140.57225.28, 225.28, 96.94
α, β, γ (o)90, 90, 9090, 90, 9090, 90, 9090, 90, 90
Mosaicity (o)0.540.20.380.17
Total no. of reflections279,204 (42,196)246,551 (36,406)185,745 (27,383)1,101,703 (164,545)
No. of unique reflections19,950 (2963)18,756 (2690)14,272 (2025)29,500 (4217)
Completeness (%)95.4 (98.2)99.8 (99.6)99.7 (99.9)100 (100)
Multiplicity14.0 (14.2)13.1 (13.5)13.0 (13.5)37.3 (39.0)
Mean I/σ(I)14.3 (1.9)16.1 (2.1)9.6 (1.6)18.0 (2.4)
Rp.i.m.0.037 (0.498)0.030 (0.455)0.049 (0.618)0.031 (0.396)
CC1/20.999 (0.767)0.999 (0.934)0.998 (0.782)0.999 (0.840)
Wilson B-factor (Å2)86.8100.111898.9
Refinement
Resolution range (Å)42.72–3.00 (3.29–3.00)40.00–3.10 (3.39–3.10)45.45–3.40 (3.72–3.40)48.77–3.00 (3.29–3.00)
No. of reflections19,89817,72013,54327,943
Rwork/Rfree0.236/0.2750.224/0.2490.271/0.2860.239/0.258
No. of non-hydrogen atoms5359538153595191
 Protein4634466846684640
 DNA713713691527
 Water1224
Average B factors (Å2)
 Protein115.3139.6165.6128.9
 DNA116.7141.2178.580.1
 Water63.281.2
R.m.s. deviations
 Bond length (Å)0.0120.0110.0100.011
 Bond angles (o)1.781.8661.7351.887
Ramachandran plot (%)
 Favored9695.2493.5493.15
 Allowed44.765.786.85
 Outliers0.68
  1. The numbers in parentheses are for the highest resolution shell. One crystal was used for each data collection.

The overall structures of p52:p52 in complex with the natural PSel-κB DNA and two variants are similar to each other with similar buried surface area and number of hydrogen bonds (H-bonds) (Figure 1D and H; Supplementary file 2). It should be noted that all the complex structures are at ~3.0 Å resolution which places limits on the identification of some detailed interactions, including hydrogen bonds. However, compared to previously known structures of NF-κB-DNA complexes, two striking differences are observed. One is that all three PSel-κB DNAs exhibited a distinct widening of the minor groove at the two base-steps around the central position 0 (−1 to 0 and 0 to +1), with width of ~7.5 Å (Figure 1E–G1). In comparison, the A/T-centric κB DNAs studied earlier, κB-33 (5′-GGAAATTTCC-3′) (Chen et al., 1998a; Huang et al., 2005) and another one that we now name κB-55 (5′-GGGAATTCCC-3′) (Moorthy et al., 2007; Fusco et al., 2009), have significantly compressed minor groove in both their bound and free states as compared to an ideal B-form DNA (Figure 1I; Figure 1—figure supplement 2A–B). Compressed minor groove width is a common feature of all A/T-centric κB DNAs bound to NF-κB dimers which is remarkably different from the minor groove width of the PSel-κB DNAs seen in the present structures (Figure 1—figure supplement 2E).

The widened minor groove is observed with long p52 proteins

The other difference observed for the three p52:p52 structures reported here concerns the organization of the dimer and the complex with DNA. The p52-MHC-κB DNA (which is A/T-centric) complex is the only previously determined crystal structure of NF-κB p52:p52 homodimer (Cramer et al., 1997). Superposition of the p52:p52 homodimer in the MHC-κB and natural PSel-κB complexes aligned by the DDs reveals large rigid body movement of NTDs with rotation of ~20° and translation along rotation axis of ~1.4 Å (Figure 2A). This results in shifting of the NTD along PSel DNA toward its flanks by ~13 Å for both sides. In addition, the minor groove of the MHC-κB DNA at the central segment is compressed like all other NF-κB-DNA complexes indicated above (Figure 1I; Figure 1—figure supplement 2C).

p52:p52 dimer conformations.

(A) (Left) Overlay of p52:p52 (aa 1–398) in complex with the 18 bp natural G/C-centric PSel-κB DNA (PDB 7CLI, this study) (in orange and green for monomers I and II, respectively) and p52:p52 (aa 35–329) in complex with the MHC-κB DNA (PDB 1A3Q) (in gray). Diagram explains rigid-body movement of the NTD. (Right) View of the complex after rotating 90° along the horizontal axis. Both protein structures are presented as backbone traces. (B) Overlay of the short p52:p52 (aa 1–327) in complex with the 13 bp PSel (mutant A/T-centric)-κB DNA (PDB 7W7L, this study) (in yellow orange and purple blue for monomers I and II, respectively) and p52:p52 (aa 35–329) in complex with the MHC-κB DNA (PDB 1A3Q) (in gray).

The PSel-κB DNAs (18 bp) and recombinant p52 protein (aa 1–398, including the GRR) used in the current study are both longer than those in the MHC-κB DNA complex (13 bp and aa 35–329). In fact, all currently available structures of NF-κB-κB DNA complexes in the Protein Data Bank (PDB) contain short NF-κB proteins (only NTD and DD) and A/T-centric κB DNAs (Figure 1—figure supplement 2E; Ghosh et al., 1995; Müller et al., 1995; Cramer et al., 1997; Chen et al., 1998a; Huang et al., 2001; Moorthy et al., 2007; Fusco et al., 2009; Chen et al., 1998b). To test if the DNA and protein length variations induce structural changes in the complex, we set out to co-crystallizations of a shorter p52 protein (aa 1–327) with all three PSel-κB DNAs at various lengths (Supplementary file 1). This short p52 protein only co-crystallized with a 13 bp PSel (mutant A/T-centric) DNA but not the (natural G/C-centric) or (−1/+1 swap) DNAs in any lengths. The conformation of this complex is nearly identical to p52-MHC-κB complex with minor groove width less than 4 Å at the central position (Figure 2B; Figure 1I; Figure 1—figure supplement 2D). This crystal with short p52 is in a different crystal form compared to the three structures with the long p52, and it is also in a different crystal form compared to the MHC-κB DNA complex, suggesting that crystal packing is unlikely to be the main cause of the structural differences, and that both the DNA and protein lengths play significant roles.

Therefore, we observed an influence of the length of the p52 protein on the conformation of the κB DNA and the organization of the p52:p52 dimer in the complex. The length and sequence of DNA also influence the structures, and we believe they are correlated with the length of p52 protein. As discussed above, the short p52 protein (aa 1–327) failed to interact with Bcl3 (Figure 1—figure supplement 1E–H), partly due to the lack of the GRR. We used the long p52 protein (aa 1–398) for the rest of the studies.

Distinct protein-DNA interactions in the (p52:p52)-DNA complexes

The widening of the minor groove propagates from the central position to all four base steps on both sides with values around 5–6 Å (Figure 1I). This widening and the consequent deepening of the major groove have significant impact on protein-DNA interactions. The most significant of which is the loss of cross-strand base contacts by Arg52 (Figure 3A). The cross-strand contacts between the homologous Arg (Arg54 in p50 and Arg33 in RelA) and DNA are observed in all other A/T-centric NF-κB-DNA structures (Figure 3B; Supplementary file 3). In the 18 bp long PSel-κB DNA complexes, the NH1 and NH2 groups of Arg52 form H-bonds with both O6 and N7 groups of G at ±3. The same Arg52 in the complex with the short 13 bp PSel (mutant A/T-centric) or the MHC-κB DNA only hydrogen bonds with the O6 group, but not the N7, of G at ±3. The homologous Arg54 in p50 also contacts the O6 group of G at −3 in the p50:RelA-IFNβ-κB DNA complex.

Figure 3 with 1 supplement see all
Protein-DNA contacts.

(A) Arg52 of p52 in the PSel-κB complex (PDB 7CLI, this study) only makes base-specific contacts with G at +3 position. (B) The corresponding Arg54 of p50 in the p50:RelA-IFNb-κB complex (PDB 1LE5) makes base-specific contacts with A at −2 and G at −3 positions as well as additional cross-strand contacts with T at −2 position. (C) Conformational differences between two p52 monomers in complex with the 18 bp natural G/C-centric PSel-κB DNA (PDB 7CLI, this study) are shown by superposing their DDs. The two monomers are presented as backbone traces. (D) Hydrogen bonding network at the interdomain interface between DD and NTD in each p52 monomer. In monomer I, the two domains form contacts with each other through a wide network of H-bonds between the side chains of Arg49 from the NTD and Gly224, Ser226, Arg311, and Asp316 from the DD; whereas in monomer II, there are only contacts between Arg49 and Ala225, as well as Ser226 and Arg311. Residues from NTD are colored in lighter orange and lighter green for monomers I and II, respectively. (E) DNA based-specific contacts made by Arg52, Arg54, His62, and Lys221 of p52 (Left) monomer I and (Right) monomer II in complex with the natural PSel-κB DNA. H-bonds are indicated as red dotted lines with distances labeled. Noted that Lys221 in monomer II is in a different conformation and has no specific contacts with DNA. (F) DNA backbone contacts made by Lys143 of p52 (Left) monomer I and (Right) monomer II. (G) Sequence alignment showing the unique basic segment in p52 among all NF-κB family members. Both human and mouse sequences of the p52 subunit are shown. Only human sequences are shown for the rest of the family members. Secondary structures and connecting loops are drawn above the sequences. (H) (Left) The unique basic segment in p52 NTD helix α2 interacts with PSel-κB DNA in the present structure (PDB 7CLI, this study); (Right) these interactions are absent in p50 subunit in the p50:RelA-IFNb-κB complex (PDB 1LE5).

The other notable feature of the PSel-κB DNA complexes is the highly asymmetric DNA contacts by p52:p52 homodimer. Monomer I is closer to its cognate half-site making more direct contacts with the DNA than monomer II (Figure 3—figure supplement 1A). Although asymmetric DNA binding by the symmetric homodimer is a common feature in all NF-κB-DNA complexes, it is significantly more pronounced in the present structures. Moreover, the p52:52 homodimer also displays substantial asymmetry. With the DD of the two monomers in superposition, the NTDs rotate from each other by ~6° (Figure 3C). The interdomain interaction is extensive in monomer I compared to that in monomer II (Figure 3D).

In the PSel-κB complex, the side chains of Lys221, Arg52, Arg54, and His62 in p52 monomer I make direct base-specific contacts to four consecutive G(s) from +2 to +5 positions (Figure 3E, left). In addition, Ser61 also makes direct contact with A at ±6 and ±7 positions; these contacts are not possible for the short p52 (aa 1–327) co-crystallized with 13 bp κB DNAs such as MHC and PSel (mutant A/T-centric)-κB (Figure 3—figure supplement 1A–B). p52 monomer II makes contact with only three G(s) from position −3 to −5 (Figure 3E, right). The conformation of loop L3 in the two p52 monomers is different; consequently, only Lys221 in monomer I makes specific contacts with G at position +2 (Figure 3—figure supplement 1C). Glu58 helps to position Arg52, Arg54, and His62, and makes base-specific interaction to the opposite C at ±3 positions.

In addition to base-specific interactions, there are multiple protein contacts to the DNA phosphate backbone, mostly to the central region of the DNA. The side chain of Cys57 hydrogen bonds to the backbone phosphate group of C at ±2; and the side chains of Tyr55 and Lys143 hydrogen bond to the backbone phosphate group of A at ±1 (Figure 3—figure supplement 1A). Only in monomer II does the side chains of Lys143 make an additional H-bond to the backbone phosphate group of C at position 0 (Figure 3F). Interestingly, all other NF-κB-DNA complexes, including the short p52:p52 homodimer bound to both 13 bp MHC-κB and PSel (mutant A/T-centric)-κB DNAs, exhibit more backbone contacts by Gln284 and Gln254 (Figure 3—figure supplement 1B; Supplementary file 3). The presence of an additional positively charged residue in loop L2 in the other NF-κB subunits (p52: T142KKN; p50: TKKK; and RelA: KKRD) enhances backbone binding at the minor groove side including cross-strand interactions (Figure 3G). In addition, there is a unique basic segment in p52, a peptide-rich in basic residues (K179ELKK), located near the end of helix α2 (Figure 3G). These basic residues possibly mediate long-range electrostatic interactions with the negatively charged DNA backbone which might pull the DNA strands away from each other toward the p52 protein (Figure 3H). In summary, aa composition in loop L2 and helix α2, might play an important role in determining DNA binding by the NF-κB dimers.

MD simulations reveal free DNAs exhibit distinct preferred conformations

In order to investigate whether the minor groove width of the PSel-κB DNA variants observed in the current complexes is induced by the protein or intrinsic to DNA sequences, we first carried out microsecond MD simulations of the four κB DNAs in free form. The simulations were initiated using DNA conformations in the crystal structures of the complexes, where the three PSel-κB DNA variants had a widened minor groove and the MHC-κB DNA a narrow minor groove. Throughout our simulations PSel (natural G/C-centric) largely maintained its widened minor groove at the central 0 position, while the minor groove in PSel (mutant A/T-centric) narrowed slightly; PSel (−1/+1 swap) displayed a significantly narrowed minor groove, which became similar to that of MHC-κB DNA recorded in the simulations (Figure 4A). The swap of T and A at ±1 positions reverses the geometric conformation of bps at both positions (Figure 4C). Specifically, these swaps exchange pyrimidines and purines at ±1 positions, forcing the swapped bps to adopt an opposite shear and buckle direction to optimize base stacking with neighboring bps compared to those on the non-swapped DNAs. The thymines at both positions slide and tilt toward the minor groove simultaneously, narrowing the central minor grooves (Figure 4—figure supplement 1A).

Figure 4 with 2 supplements see all
MD simulations for free κB DNAs.

(A, B) Statistics of the minor groove width over the aggregated 10-µs simulations of each system at (A) the central 0 position (averaged over the five levels from −1 to +1 positions) and (B) the +1 position (averaged over the five levels from 0 to +2 positions). (Upper) DNA isosurface at 0.2 isovalue (20% occupancy); (Lower) Probability distribution of minor groove width. Dashed lines show the minor groove width in the (p52:p52)-bound crystal structures. (C) Representative structures of natural G/C-centric, mutant A/T-centric, −1/+1 swap PSel-κB DNAs, and MHC-κB DNA revealed from MD simulations. (Left) Superimposed structures showing the narrowed central minor groove on −1/+1 swap DNAs; (Right) Representative conformations of bps at −1, 0, and +1 positions revealed from MD simulations. Minor groove width was calculated with Curves+ (Blanchet et al., 2011; Lavery et al., 2009). MD, molecular dynamics.

The simulations also reveal a narrowed minor groove of the A/T-centric DNA at +1 position compared to the corresponding G/C-centric DNA with the same flanking bps, that is, PSel (mutant A/T-centric) compared to PSel (natural G/C-centric), and MHC compared to PSel (−1/+1 swap) (Figure 4B). The A:T bp at 0 position shows large shear, buckle, and opening, forcing a register toward the minor groove in the curvature of free A/T-centric DNAs (Figure 4—figure supplement 1A). With only two H-bonds, A/T steps can easily unwind to yield low propeller twist. The neighboring A:T at 0 and +1 position in PSel (mutant A/T-centric) (0 and −1 position in MHC) rotate the adenines likewise to optimize base stacking (Figure 4C). This conformation bends the minor groove and can further deform the local structure of PSel (mutant A/T-centric) through the occasionally formed cross-strand H-bonds between the neighboring steps (Figure 4—figure supplement 2). It appears that in MHC, the same narrowing effect introduced by ±1 swap fixed the twisted conformation at 0 position, thereby stabilizing its local structure instead. Collectively, we conclude that having successive A:T is likely to reduce the intrinsic minor groove width. Our finding is in line with the observations of a compressed minor groove of free κB-33 DNA or the bending into the minor groove from continuous A:T in A-tract DNAs (Barbic et al., 2003; Huang et al., 2005).

Comparison of MD simulations and crystal structures suggests that upon binding to p52, the −1/+1 swap DNA experiences more disruptive conformational change than the natural G/C-centric PSel-κB (Figure 4A; Figure 4—figure supplement 1B). The binding at the central part of both DNAs is symmetrically facilitated through the H-bonds between two residues (Lys143 and Tyr55) from each monomer of p52 and the phosphate of nucleotides at −1 and +1 positions as well as the T-shaped π-stacking interactions between DNA bases and Tyr55. The aforementioned two residues in the two p52 monomers are positioned further apart in the PSel-κB DNA complex compared to the MHC-κB DNA complex. Unlike the natural G/C-centric DNA, the binding on both strands of −1/+1 swap DNA draws the bound thymine in the opposite directions of the minor groove, breaking the intra-bp H-bonds and severely distorting the central bps (Figure 4—figure supplement 1B). It appears that p52:p52 homodimer adopts a specific conformation with a nearly fixed inter-monomer distance upon the binding to κB DNAs, such that it tears the central part of −1/+1 swap DNA into a favored binding conformation. Overall, the comparative analysis of MD simulations and crystal structures suggests that the p52:p52 homodimer induces the least amount of conformational changes on κB DNA with an intrinsically widened minor groove.

The p52 homodimer recognizes κB DNAs with different thermodynamic features

Structural analysis described above did not provide a strong correlation between the conformational states of (p52:p52)-DNA complexes and the transcriptional output. We next tested whether p52:p52 homodimer binds to the natural G/C-centric, mutant A/T-centric, and −1/+1 swap PSel-κB, as well as MHC-κB DNAs with different mechanisms and/or affinities. In all cases, long p52 protein (aa 1–398) as well as long DNA were used. Isothermal titration calorimetry (ITC) reveals that p52:p52 binds all three PSel-κB DNA variants with similar binding affinities (Kd) in the range of approximately 40–90 nM whereas it binds MHC-κB DNA much tighter (Figure 5; Figure 5—figure supplement 1). However, binding of p52 to the natural G/C-centric PSel-κB DNA is associated with a large increase in entropy (ΔS) and a moderate decrease in enthalpy (ΔH). On the other hand, the binding to the MHC and mutant A/T-centric PSel DNAs showed a much larger decrease in enthalpy. These results suggest that the binding of p52:p52 homodimer to the G/C-centric κB DNA is favored more by entropy, whereas the binding to the A/T-centric DNA is driven by enthalpy alone.

Figure 5 with 2 supplements see all
p52 binds κB DNAs with different thermodynamic features.

(A–D) Calorimetric titration data showing the binding of recombinant p52:p52 (aa 1–398) homodimer with (A) natural G/C-centric, (B) mutant A/T-centric, (C) −1/+1 swap PSel-κB, and (D) MHC-κB DNAs. The top panel of the ITC figures is the raw data plot of heat flow over time for the titration of 300 μM of indicated DNA into 35 µM of p52; the bottom panel shows the corresponding plot after integration of peak areas and normalization to yield a plot of ΔH against the DNA/p52 ratio and the line represents the best fit to the data according to a single-site binding model. The determined Kd, changes of enthalpy and entropy are shown on the bottom panel. ITC, isothermal titration calorimetry.

To test if this mechanism is general to other κB DNAs, we also determined the thermodynamic parameters for p52 binding to Skp2-κB DNA. Skp2-κB DNA is present in the promoter of S-phase kinase-associated protein 2 (Skp2) and it is also regulated by the p52:p52 homodimer and Bcl3 (Figure 5—figure supplement 2A; Barré and Perkins, 2007; Wang et al., 2012). Skp2-κB DNA is another natural G/C-centric (5′-GGGGAGTTCC-3′) κB DNA but with the presence of A:T and T:A bp at +1 and −1 positions, the same as the −1/+1 swap PSel DNA in the central region. Skp2 also has a very different 4 bp half-site, TTCC, at the +1 to +4 positions. The Kd as well as relative contributions of entropy and enthalpy to the binding to Skp2 and −1/+1 swap PSel DNAs are similar (Figure 5C; Figure 5—figure supplement 1C, Figure 5—figure supplement 2B). These results suggest that DNA sequence and conformational differences lead p52 to bind DNAs through different thermodynamic binding processes. However, thermodynamic binding mechanism does not fully capture the differential transcriptional output mediated by these κB DNAs.

The p52 homodimer binds κB DNAs with different kinetic features

We next examined the binding kinetics of p52 and κB DNAs as there is mounting evidence that, separate from binding affinity, kinetic rate constants (kon and koff) are crucial to the physiological effects of protein-ligand interactions in a variety of cellular processes (Nakajima et al., 2001; Gross and Lodish, 2006; González et al., 2005; Markgren et al., 2002). We utilized biolayer interferometry (BLI) to study the association and dissociation rate of p52:p52 binding to various κB DNAs. Biotinylated DNAs were immobilized on the streptavidin (SA) sensors and tested with purified p52 protein. The binding kinetics differ significantly among the DNAs. The binding of more transcriptionally active natural G/C-centric PSel showed a higher association (kon) and dissociation rate (koff) than the other two variants and MHC-κB DNAs (Figure 6A–E). Consistently, in the case of Skp2-κB DNA, the more transcriptionally active natural G/C-centric Skp2 showed a faster kinetics than its mutant A/T-centric, especially the koff (Figure 6—figure supplement 1A–C).

Figure 6 with 2 supplements see all
p52 binds the natural transcriptionally active G/C-centric PSel-κB DNA with faster kinetics.

(A–D) Biolayer interferometry (BLI) binding analysis of p52:p52 (aa 1–398) homodimer to immobilized biotin labeled (A) natural G/C-centric, (B) mutant A/T-centric, (C) −1/+1 swap PSel-κB, and (D) MHC-κB DNAs. The differences in kon and koff can be seen in the shapes of the association and dissociation curves. Each experiment was done in duplicate and one representative set of curves is shown. (E) Table showing the kinetic analysis in (A–D). (F–I) BLI binding analysis of p52:p52:Bcl3 complex to immobilized biotin labeled (F) natural G/C-centric, (G) mutant A/T-centric, (H) −1/+1 swap PSel-κB, and (I) MHC-κB DNAs. Each experiment was done in duplicate and one representative set of curves is shown. (J) Table showing the kinetic analysis in (F–I). (K) Table summarizing the fold change of Kd, kon, and koff with respect to the more transcriptionally active G/C-centric PSel-κB DNA. The average values of the duplicated kinetics data in (A–J) and the relative reporter activities in RLU from Figure 1A–B were used for ratio calculations. The numbers for the greater reporter active G/C-centric PSel are shown in blue.

We further determined the kon and koff of the transcriptionally competent p52:p52:Bcl3 complex binding to PSel-κB DNA variants by BLI. In agreement with our previous study, only the recombinant phospho-mimetic Bcl3 from Escherichia coli forms ternary complex with p52:p52 homodimer and κB DNA (Wang et al., 2017). Both recombinant WT and phospho-mimetic Bcl3 protein interact with p52 with similar kinetics (Figure 1—figure supplement 1F, Figure 6—figure supplement 2A–C); however, the p52:p52:WT-Bcl3 complex does not bind DNAs (Figure 6—figure supplement 2D–E). The binding of p52:p52:phospho-Bcl3 with the natural G/C-centric PSel DNA exhibited both higher kon and koff (Figure 6F–J). Similarly, the binding with the natural G/C-centric Skp2 DNA also showed a higher koff comparedto its A/T-centric mutant (Figure 6—figure supplement 1D–F).

Overall, the binding kinetics of p52:p52 homodimer alone versus p52:p52:Bcl3 complex follows the same trend. Moreover, a comparison of binding affinity, association, and dissociation rates with respect to the more transcriptionally active PSel and Skp2-κB sites shows a correlation between transcriptional output and the dissociation rate. The slower the koff, the lower the reporter activities for both (p52:p52)-DNA and (p52:p52:Bcl3)-DNA complexes (Figure 6K; Figure 6—figure supplement 1G). Therefore, transcriptional activity may have a closer link to the binding kinetics rather than the thermodynamic stability of the complex.

Differential minor groove geometries correlate with differential DNA binding kinetics

To better understand how binding kinetics correlates with DNA sequences, we carried out four 3-µs MD simulations for each of the three (p52:p52)-PSel-κB DNA complexes, including the natural G/C-centric, mutant A/T-centric, and −1/+1 swap DNAs. The overall conformations of p52:p52 dimers and DNAs are similar during the MD simulations, both of which are slightly more stable in the p52-natural G/C-centric DNA complex (Figure 7—figure supplement 1A–C). Upon p52:p52 homodimer binding, the central minor grooves of all three DNAs narrowed, resulting in a consistent trend as seen in the free form (Figure 7—figure supplement 1D). Adapting to the width of the DNA central minor groove, the p52:p52 subunits bound to −1/+1 swap DNA adopt a closed conformation where the two segments bound to the central minor groove appear to clamp the DNA, in contrast to their more open conformation when bound to the natural G/C-centric DNA; the p52 subunits bound to the mutant A/T-centric DNA fall in between the aforementioned two types of conformations (Figure 7—figure supplement 2). Notably, we found that the NTD of p52:p52 subunits could make contacts across the central minor grooves when bound to mutant A/T-centric and −1/+1 swap DNAs (Figure 7A). Specifically, the clamping conformation enables Lys144 to form cross-strand binding that engages the phosphates at −3/+3 positions in the opposite DNA strands in the mutant A/T and −1/+1 swap DNAs (Figure 7B). In the case of the −1/+1 swap DNA, the main chain amines of Lys144 in both monomers also retain contacts to the phosphates at −1/+1 positions in the nearby strand, likely due to the matching lysine side chain length and the narrowed minor groove width (Figure 7B–C). These cross-strand contacts, while recorded in all three protein-DNA complexes, were most frequently observed in the −1/+1 swap and mutant A/T-centric DNAs. Specifically, they were approximately five and three times more frequent than in the natural G/C-centric DNA, respectively (Figure 7D). This difference suggests that the dynamism of the p52:p52 homodimer varies as it recognizes specific minor groove geometries of the three PSel-κB DNAs. The DNA binding domains remain open preventing Lys144 from reaching out to the other DNA strand in the natural G/C-centric DNA. In the case of the −1/+1 swap DNA, the two p52 NTDs adopt a closed conformation allowing Lys144 to make cross-strand contacts which are further assisted by the DNA conformational change. On the one hand, such cross-strand binding provides extra contacts between the p52:p52 homodimer and the DNA, which may hinder their dissociation. On the other hand, the induced closing of NTDs may be unfavored by the p52:p52 homodimer and slow down the protein-DNA association.

Figure 7 with 2 supplements see all
MD simulations for (p52:p52)-DNA complexes.

(A) Isosurface of (p52:p52)-DNA complexes (20% occupancy). Red arrows indicate the cross-strand contacts by p52 homodimer observed in conformations bound to mutant A/T-centric and −1/+1 swap PSel-κB. (B) Representative binding conformations of Lys144 at the minor grooves showing the cross-strand binding formed in mutant A/T-centric and −1/+1 swap PSel-κB. Red dashed lines stand for the hydrogen bonds formed between Lys144 and DNAs. (C) Probability distributions of minimum distance between the α-N of Lys144 backbone and the P atom at position −1/+1 of the nearby strand of DNA, and (D) between the ε-N of Lys144 side chain and the P atom at position +3/–3 of the far strand of DNA. The cumulative probabilities of close contacts (<4.8 Å) are marked in the same coloring scheme. MD, molecular dynamics.

To further probe the role of Lys144 identified in the simulations, this residue was mutated to Ala (p52K144A) and assessed for binding to all three PSel-κB DNA variants. The binding kinetics of p52K144A:p52K144A homodimer alone with DNAs as well as the transcriptionally competent p52K144A:p52K144A:Bcl3 complex were tested. The p52K144A mutant was expressed and purified to similar purity as the WT p52 protein, and the mutation does not affect its interaction with Bcl3 (Figure 8—figure supplement 1A–C). Overall, the p52K144A mutant binds to all three PSel-κB DNAs with weaker affinity compared to the WT p52 (Figure 8; Figure 6), likely due to the completely abolished Lys144 cross-strand contacts. Consistent with the simulation analysis, dissociation of all three DNAs from p52K144A mutant is sped up relative to the WT p52, with the most prominent change recorded for −1/+1 swap. Meanwhile, the reduction of their kon suggests a promotion effect of Lys144 on the closing and DNA-binding of WT p52:p52 homodimer, which may arise from the favorable electrostatic interactions between this residue and the DNAs. Compared to the natural G/C-centric DNA, the −1/+1 swap DNA binds to p52K144A mutant with significantly slower kon and koff, and the difference between the two DNAs increases upon K144A mutation; the rate constants are in the middle of the aforementioned two DNAs in the mutant A/T-centric DNA. Overall, the substitution of the positively charged Lys144 by an alanine appears to slow down the recognition of the DNA minor grooves, which may in turn slow down the closing of p52K144A:p52K144A homodimer, thereby, decreasing the association rate of the DNAs especially for the ones with narrower minor grooves such as the −1/+1 swap DNA.

Figure 8 with 1 supplement see all
p52K144A binds κB DNAs with slower kinetics.

(A–C) BLI binding analysis of p52K144A:p52K144A (aa 1–398) homodimer to immobilized biotin labeled (A) natural G/C-centric, (B) mutant A/T-centric, and (C) −1/+1 swap PSel-κB DNAs. Each experiment was done in duplicate and one representative set of curves is shown. (D) Table showing the kinetic analysis in (A–C). (E–G) BLI binding analysis of p52K144A:p52K144A:Bcl3 complex to immobilized biotin labeled (E) natural G/C-centric, (F) mutant A/T-centric, and (G) −1/+1 swap PSel-κB DNAs. Each experiment was done in duplicate and one representative set of curves is shown. (H) Table showing the kinetic analysis in (E–G). BLI, biolayer interferometry.

Discussion

The p52 homodimer recognizes κB DNA using a mode distinct from other NF-κB dimers

Double-stranded DNA helices are not static entities that simply present themselves to proteins and assemble into multiprotein complexes at specific sequences. The DNA duplex is intrinsically dynamic on many levels and time scales in cells. The movement of DNA through its different conformational states is continuous and is influenced by, but not completely dependent upon, its nucleotide sequence. Structures presented in this study show that the conformations of all three PSel-κB DNA variants bound to the long p52:p52 homodimer are similar but are distinct from all previously known complexes between κB DNAs and six other NF-κB dimers (p50:p50, p50:RelA, p50:RelB, RelA:RelA, c-Rel:c-Rel, and p52:RelB). It was noted earlier a compressed minor groove in the central region of the DNA is a key feature of NF-κB-DNA complexes. The minor groove at the central three positions is significantly widened in all three complexes presented here. However, MD simulations show free DNAs exist in distinct preferred conformations, which appear to be adjusted by p52 into a unique shape for recognition. And notably, the more transcriptionally active natural PSel-κB DNA appears to maintain similar conformational and dynamic states in free and bound forms.

The current structures also demonstrate a correlation between the p52 protein length and the conformation of the κB DNA. The GRR region of the p52 protein, which was not included in any previous NF-κB structural studies, seems to play an important role. However, no electron density was observed for the GRR region in the current structures. Future studies are needed to fully understand the role of the GRR region in (p52:p52)-DNA complex conformation and the interaction with cofactor Bcl3.

Binding affinity does not fully capture the transcriptional activity

To determine if binding affinity is related to transcriptional activity, we measured the affinity of all complexes under equilibrium condition. Surprisingly, but consistent with our previous report (Mulero et al., 2018), we found no correlation between affinity and transcriptional activity. The p52:p52 homodimer binds to MHC-κB with the highest affinity but it is not a transcriptional activation competent complex. Interestingly, our analysis reveals that p52:p52 homodimer uses different paths to bind κB DNAs ranging from a more entropic favored for the natural PSel, to exclusively enthalpic for MHC, and to mixed entropic-enthalpic for mutant A/T-centric and −1/+1 swap PSel DNAs. The entropy-favored processes are linked to faster binding kinetics: p52:p52 homodimer binds to natural G/C-centric PSel DNA with both faster association and dissociation rates. The most populated conformation of the free G/C-centric PSel DNA as revealed by MD simulation is similar to the one observed in the crystal structure of the complex suggesting this DNA’s conformation does not undergo significant changes upon protein binding. Thus, in the complex between p52:p52 and natural G/C-centric DNA, both the DNA and protein most likely preserve their native states. This could account for the positive entropy and faster kon. However, possibly the protein-DNA contacts in such a complex are suboptimal resulting in their faster dissociation. In contrast, p52:p52 complexes with the mutant A/T-centric or−1/+1 swap DNAs likely involve rigidification of protein-DNA contacts, requiring some structural reorganizations in both molecules, resulting in more enthalpically stable complexes and slower association and dissociation rates. Indeed, MD simulations of the (p52:p52)-DNA complexes are consistent with this notion. The complex of p52:p52 homodimer with the natural G/C-centric PSel-κB DNA maintains very similar conformations under simulations as seen in the crystal structure. In contrast, induced by their narrowed minor groove geometries, the p52 protein closes on the DNAs in the PSel (−1/+1 swap) complex and to a lesser extent in the (mutant-A/T-centric) complex, which is likely unfavored by the p52:p52 homodimer conformation. These structural changes led to the cross-strand contacts by Lys144 in the −1/+1 swap and mutant A/T-centric DNAs but not in the natural G/C-centric PSel DNA. Combination of MD simulations and structural studies supports the model as PSel (mutant A/T-centric) and (−1/+1 swap) DNAs undergo conformational changes from free to bound states. Our observations are consistent with other studies which have shown that rapid association and dissociation are favored by entropy, whereas slow association and dissociation are guided by enthalpy (Baerga-Ortiz et al., 2004).

Ideas and speculation: transcriptional regulation via kinetic discrimination

Understanding transcriptional regulation has attracted many researchers since the discovery of the lac operon. One of the most intriguing questions scientists are working to resolve is the mechanism of transcriptional regulation by the specific DNA response elements. Affinity regulation by different target DNA sequences for a TF has long been thought to be the dominant mode of regulation imposed by such DNA sequences. Indeed, in many cases of eukaryotic transcription differential affinity has been shown to be critical (Sekiya et al., 2009). TFs are also known to bind free DNA or nucleosome with distinct kinetics (Donovan et al., 2019). But none of these studies has established a direct correlation between binding kinetics and transcriptional regulation in eukaryotes. Many biological systems have been studied in detail with the roles of binding kinetics in regulation evaluated. For instance, receptor-ligand interactions, T cell activation, and potency of bacterial toxins are guided by the half-life of key complexes (Corzo, 2006; González et al., 2005; Gross and Lodish, 2006; Nakajima et al., 2001).

Of all six κB DNAs tested, the natural G/C-centric PSel and Skp2 DNAs showed greater transcriptional activation. We found that a slower dissociation rate or longer residence time is linked to reduced transcriptional activation. Although the relationship between the dissociation rates and transcription activities is shown for only six tested binding sites, this relationship is preserved for (p52:p52)-DNA complexes and to a lesser extent for (p52:p52:Bcl3)-DNA complexes. The relationship between binding kinetics and conformations of the complexes is further supported by the study of a mutant. This mutant, p52K144A, binds all DNAs with a slower kinetics but the kinetics is the most prominently slowed down for the PSel (−1/+1 swap) DNA.

The rate constants obtained in our in vitro assays probably are not the same in vivo, where many other factors will have an impact on binding kinetics. However, the relative rates clearly suggest that the persistent presence of p52 on DNA gives rise to less transcription. Work presented here hints at a link between the DNA binding kinetics of a TF and its interaction with coactivators and corepressors. We previously showed that the p52:p52:Bcl3 complex preferentially recruits HDAC3 when it remains bound to an A/T-centric κB site (Wang et al., 2012), it is possible that the slower dissociation rate or longer residence time of p52:p52:Bcl3 on the A/T-centric κB site described in this study matches the slower on rate of the corepressor to p52:p52:Bcl3 bound to A/T DNA. That is, the A/T-centric κB DNA:p52:p52:Bcl3 complex remains stable for long enough to give the HDAC3 corepressor complex enough time to stably interact with it. In addition, the binding of other TFs to the promoters/enhancers of target genes inevitably impacts on the coactivator/corepressor regulation by the (p52:p52)-DNA complexes. Future experiments aimed at coactivator and corepressor interaction rates within the context of chromatinized DNA are needed to verify the validity of the kinetic model for DNA element sequence-specific gene regulation.

In summary, our studies have revealed a novel conformation for κB DNA in complex with NF-κB and a new organization of an NF-κB dimer. More importantly, our work provides a new insight into the mechanism of differential thermodynamics and kinetics of NF-κB-DNA binding. DNA response elements with only 1 or 2 bp variations could provoke drastically different kinetic and thermodynamic effects. Future experiments will help us fully understand how such binding processes result in transcription activation or repression.

Materials and methods

Protein expression and purification

Recombinant non-tagged human p52 (aa 1–398), (aa 1–398) K144A mutant, and (aa 1–327) was expressed and purified from E. coli Rosetta (DE3) cells. Rosetta (DE3) cells transformed with pET-11a-p52 (aa 1–398), (aa 1–398) K144A, or (aa 1–327) were cultured in 2 L of LB medium containing 50 mg/mL ampicillin and 34 mg/mL chloramphenicol at 37°C. Expression was induced with 0.2 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at OD600 0.5–0.6 for 3 hr. Cells were harvested by centrifugation, suspended in 40 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM β-Mercaptoethanol (β-ME), 1 mM PMSF, and lysed by sonication. Cell debris was removed by centrifugation (20,000×g for 30 min). Clarified supernatant was loaded onto Q-Sepharose FF column (GE Healthcare). Flow through fraction was applied to SP HP column (GE Healthcare). The column was washed with 40 mM Tris-HCl (pH 7.5), 200 mM NaCl, 10 mM β-ME, and the protein was eluted by the same buffer containing 400 mM NaCl. p52 was concentrated and loaded onto the gel filtration column (HiLoad 16/600 Superdex 200 pg, GE Healthcare) pre-equilibrated with 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 5 mM β-ME. Peak fractions were concentrated to ~10 mg/mL, flash frozen in liquid nitrogen, and stored at –80°C.

His-Bcl3 (1-446) WT and phospho-mimetic mutant was expressed in E. coli Rosetta (DE3) cells by induction with 0.2 mM IPTG at OD600 0.4 for 8 hr at 24°C. Cell pellets of 2 L culture of Bcl3 alone or together with 1 L culture of p52 (for p52:p52:Bcl3 complex) were resuspended together in buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 25 mM imidazole, 10% glycerol, 10 mM β-ME, 0.1 mM PMSF, and 50 μL protease inhibitor cocktail (Sigma-Aldrich) and then purified by Ni Sepharose (HisTrap HP, GE Healthcare) followed by anion exchange column (Q Sepharose fast flow, GE Healthcare). The protein complex further went through HiTrap Desalting Column (GE Healthcare) to exchange buffer before BLI assays.

Crystallization, data collection, and structure determination

Annealed DNA duplex was mixed in 20% molar excess with the pure protein.

The crystals of the p52 (aa 1–398):PSel(G/C-centric) 18 bp complex were obtained by the sitting-drop vapor diffusion method at 20°C with a reservoir solution containing 0.1 M sodium malonate (pH 4.0), 0.2 M CsCl, and 5% (w/v) PEG 3350.

The crystals of the p52 (aa 1–398):PSel(A/T-centric) 18 bp complex and the p52(aa 1–398):PSel (−1/+1 swap) 18 bp complex were obtained by the sitting-drop vapor diffusion method at 20°C with a reservoir solution containing 0.1 M sodium malonate (pH 4.0), 50 mM CsCl, and 2.5% (w/v) PEG 3350.

The crystals of the p52 (aa 1–327):Psel(A/T-centric) 13 bp complex were obtained by the sitting-drop vapor diffusion method at 20°C with a reservoir solution containing 50 mM MES (pH 6.0), 10 mM MgCl2, and 10% (w/v) PEG 3350.

Before data collection, all crystals were briefly soaked in their original crystallization solution with 20% (v/v) ethylene glycol. All crystals were flash frozen in liquid nitrogen for diffraction screening and data collection at 100K. X-ray diffraction data were collected at beamline BL19U1 at Shanghai Synchrotron Radiation Facility. The initial solution was obtained by molecular replacement using Phaser (RRID:SCR_014219) (McCoy et al., 2007) with p52-MHC DNA complex (Cramer et al., 1997) as the search model. The structure was further refined through an iterative combination of refinement with Refmac5 (RRID:SCR_014225) (Murshudov et al., 2011) and manual building in the Coot program (RRID:SCR_014222) (Emsley and Cowtan, 2004; Emsley et al., 2010). The crystallographic information is summarized in Table 1.

MD simulation

All free DNA MD simulations were carried out in GROMACS 2020.6 (Abraham et al., 2015) with Amber14sb force field (Maier et al., 2015) and OL15 parameters for DNA (Zgarbová et al., 2015), whereas all (p52:p52)-DNA complex MD simulations were conducted in GROMACS 2021.4 with Amber19sb force field (Tian et al., 2020) and OL15 parameters for DNA. To prepare the free DNA systems, crystal structures of κB/κB-like DNAs were extracted from the corresponding experimentally resolved p52:p52 dimer-bound structures, whereas the MHC DNA was retrieved from RCSB PDB database (RRID:SCR_012820) (PDB 1A3Q) (Cramer et al., 1997). In each system, κB DNA or (p52:p52)-DNA complex was placed in the center of a dodecahedron box with a 12 Å margin, solvated with TIP3P water (Jorgensen et al., 1983), and ionized with 0.1 M NaCl. Energy minimization was performed until the maximum force of system was below 1000 kJ·mol–1·nm–1. The minimized system was then equilibrated in a NVT ensemble for two 1-ns stages, where the DNA heavy atoms were harmonically restrained with a force constant of 20,000 and 10,000 kJ·mol–1·nm–2, respectively. Subsequently, the system was subjected to a 6-ns position-restrained NPT equilibration, with the force constant of DNA restraint gradually reduced from 10,000 to 400 kJ·mol–1·nm–2. At the meantime, the protein heavy atoms were harmonically restrained with a force constant of 1000 kJ·mol–1·nm–2 throughout the NVT and NPT equilibrations. Finally, five replicas of 2-µs unrestrained production simulations were performed for each DNA system, resulting in an aggregated 10-µs trajectory for each free DNA system and 12-µs trajectory for each (p52:p52)-DNA complex system, with a total simulation time of 76 µs. In all simulations, van der Waals forces were smoothly switched to 0 from 9 to 10 Å. Electrostatics were calculated using the particle mesh Ewald (PME) method (Darden et al., 1993) with a cutoff of 10 Å. A velocity-rescaling thermostat (Bussi et al., 2007) was employed for the temperature coupling at 300K, whereas pressure coupling at 1 bar was implemented by a Berendsen barostat (Berendsen et al., 1984). All bonds involving H atoms were constrained using the LINCS algorithm (Hess et al., 1997).

Occupancy of DNA and p52:p52 homodimer were calculated over the corresponding integrated trajectories using the VolMap tool in visual molecular dynamics (VMD) (RRID:SCR_001820) (Humphrey et al., 1996). The clustering analyses were conducted within GROMACS packages (RRID:SCR_014565) using GROMOS method. Representative structures or conformations were obtained from the centroid structures of top clusters from the clustering analysis of the corresponding structural elements and rendered with PyMOL (RRID:SCR_000305) (Schrodinger, 2015). The hydrogen bonds were calculated using PyMOL with default standard (heavy atom distance cutoff of 3.6 Å and angle cutoff of 63°). The bp and groove parameters were measured via Curves+ (RRID:SCR_023268) (Lavery et al., 2009; Blanchet et al., 2011), with the uncertainty represented by the standard error of the mean computed from the five replica simulations of a given system.

ITC assays

ITC measurements were carried out on a MicroCal iTC200 (Malvern Inc) at 25°C. The ITC protein sample p52 (1–398) went through desalting column (HiTrap desalting, GE Healthcare) to freshly made ITC buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 1 mM dithiothreitol (DTT). DNA oligos were dissolved in the same freshly made ITC buffer; equal amount of top and bottom strands of the oligos were mixed followed by heating at 95°C for 10 min and then slowly cooling down to room temperature for annealing. About 35 µM p52 (1–398) protein (in cell) was titrated with 300 µM DNAs (in syringe). A time interval of 150 sbetween injections was used to ensure that the titration peak returned to the baseline. The titration data were analyzed using the program Origin7.0 (RRID:SCR_014212) and fitted by the One Set of Site model.

BLI assays

The kinetic assays were performed on Octet K2 (ForteBio) instrument at 20°C with shaking at 1000 RPM. The SA biosensors were used for protein-DNA interactions and were hydrated in BLI buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM DTT, and 0.02% (v/v) Tween-20. All DNAs used were 20-mer in length and biotin-triethyleneglycol (TEG) labeled. The DNAs were loaded at 50 nM for 300 s prior to baseline equilibration for 60 s in the BLI buffer. Association of p52:p52 (aa 1–398) or p52:p52:Bcl3 complex in BLI buffer at various concentrations was carried out for 400 s prior to dissociation for 600 s. The Ni-NTA biosensor was used for protein-protein interactions and was hydrated in BLI buffer containing 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, 5% glycerol, 1 mM DTT, and 0.02% (v/v) Tween-20. His-tagged-Bcl3 was loaded at 500 μg/mL for 90 s prior to baseline equilibration for 180 s in the BLI buffer. The association of p52 in BLI buffer at various concentrations was carried out for 240 s prior to dissociation for 360 s. All data were baseline subtracted and analyzed Sartorius Octet BLI analysis system (RRID:SCR_023267) using a global fitting to a 1:1 binding model. The experiments were done in duplicate.

Luciferase reporter assays

HeLa cells were obtained from ATCC (CCL-2) (RRID:CVCL_0030). Cell cultures were tested negative for mycoplasma infection (Universal Mycoplasma Detection kit 30–1012K, ATCC). HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, catalog #11995065) that was supplemented with 10% fetal bovine serum (FBS; Gibco, catalog #10270106) and 1× Penicillin-Streptomycin-L-Glutamine (Corning, catalog #30-009Cl). HeLa cells were transiently transfected with Flag-p52 (1–415) together with Flag-Bcl3 (1–446) expression vectors or empty Flag-vector, and the luciferase reporter DNA with specific κB DNA promoter (Wang et al., 2012). The total amount of plasmid DNA was kept constant for all assays. Transient transfections were carried out using Lipofectamine 2000 (Invitrogen). Cells were collected 48 hr after transfection. Luciferase activity assays were performed using Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s protocol. Data are represented as mean standard deviations (SDs) of three independent experiments in triplicates.

Data availability

The atomic coordinates have been deposited in the Protein Data Bank, https://www.wwpdb.org/ (PDB ID codes 7CLI, 7VUQ, 7VUP and 7W7L).

The following data sets were generated
    1. Meshcheryakov VA
    2. Wang VY-F
    (2023) RCSB Protein Data Bank
    ID 7CLI. Structure of NF-kB p52 homodimer bound to P-Selectin kB DNA fragment.
    1. Meshcheryakov VA
    2. Wang VY-F
    (2023) RCSB Protein Data Bank
    ID 7VUQ. Structure of NF-kB p52 homodimer bound to A/T-centric P-Selectin kB DNA fragment.
    1. Meshcheryakov VA
    2. Wang VY-F
    (2023) RCSB Protein Data Bank
    ID 7VUP. Structure of NF-kB p52 homodimer bound to +1/-1 swap P-Selectin kB DNA fragment.
    1. Meshcheryakov VA
    2. Wang VY-F
    (2023) RCSB Protein Data Bank
    ID 7W7L. Structure of NF-kB p52 homodimer bound to 13-mer A/T-centric P-Selectin kB DNA fragment.

References

    1. Darden T
    2. York D
    3. Pedersen L
    (1993)
    Particle mesh ewald - an N.log(N) method for ewald sums in large systems
    Journal of Chemical Physics 98:10089–10092.
    1. Emsley P
    2. Cowtan K
    (2004) Coot: model-building tools for molecular graphics
    Acta Crystallographica. Section D, Biological Crystallography 60:2126–2132.
    https://doi.org/10.1107/S0907444904019158
    1. Emsley P
    2. Lohkamp B
    3. Scott WG
    4. Cowtan K
    (2010) Features and development of coot
    Acta Crystallographica. Section D, Biological Crystallography 66:486–501.
    https://doi.org/10.1107/S0907444910007493

Decision letter

  1. Volker Dötsch
    Senior and Reviewing Editor; Goethe University, Germany

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your article "Structures of NF-κB p52 homodimer-DNA complexes rationalize binding mechanisms and transcription activation" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Volker Dötsch as the Reviewing Editor and Reviewer #3, and the evaluation has been overseen by a Reviewing Editor and Volker Dötsch as the Senior Editor.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) The resolution obtained for the crystal structures does not allow the identification of individual hydrogen bonds and other details. These limitations should be discussed since these details are important for the structural interpretation.

2) The data shown in Figure 1 indicate a wider minor groove for p52 homodimers with C-terminal extensions bound to long (18bp) kB sites (PSel, PSelG->A, pSelAT) and a narrower minor groove for minimal p52 homodimers bound to 13 bp kB sites (pSelAT, MHC). The authors suggest that the protein length may be important, but they do not discuss that the DNA length may also influence the structures. The same kB sequence (PSelAT) appears in both compressed and expanded minor groove forms. Analysis of the structures indicates that contacts (Ser61-A6,A7) are made outside of the 11-bp kB site in the 18-bp structures and would not be present in the 13-bp structures. So, the conclusion that p52 bound to PSel kB has a widened minor groove relative to other complexes may be correct, but it is far from solid given the data that is presented.

3) The main question of the manuscript is what is different about p52-PSel kB contacts compared to kB sites with a central A/T (like MHC)? The overall goal is to understand how a single bp can make such a large difference in the transcriptional outcome, but the answer is not clear. Loss of a cross-strand interaction is discussed, but a new Arg-Gua interaction is gained and that is not discussed. There is a lot of detail about the asymmetric nature of the complex, but most of that appears to be common to all kB sites. A better definition and discussion of the structural source of the observed selectivity is necessary.

4) It is not obvious how the raw ITC data are converted into the integrated data shown below the raw data. In principle, the dilution heat has to be subtracted followed by the integration of the peak. In the graph with the integrated data, the first 7 points in A have the same value (as is expected for such a strong binding). But the peaks in the raw data diagram vary substantially. That could be due to differences in the width of the peaks, but that should be explained as why it happens and shown. It would also help to have at least one repetition of the experiments for validation purposes.

5) Related: How was the DNA prepared for the ITC measurements? In two of the four diagrams showing the raw data substantial dilution heat of the DNA is measured. Since the DNA sequence is very similar in all cases, a similar dilution heat is expected. Is there any impurity in two of the four preparations (that would also influence the overall results)?

6) Adding an error estimate for ΔS would allow readers to evaluate whether the differences are significant. There is an amazing 3-fold variation in ΔH, from -5 kcal/mol to -15 kcal/mol. Equally amazing is that the entropic contribution TΔS also varies widely, from +5 kcal/mol to -5 kcal/mol. I found it surprising that there was no attempt to relate these findings to the known structures. Do the number of polar interactions differ? Solvent accessible surface? Amount of ordered solvent?

7) The Kd values determined from the kinetic measurements differ by > 50-fold from the calorimetry results. This discrepancy needs to be addressed. Can the on and off-rates be trusted in these experiments? Even if they are relative rates, one might think that their ratios (K) would still be relevant.

8) None of the binding events are 'entropy driven'. The most favorable (PSel kB) is still 50:50 enthalpy:entropy.

9) Why would binding with native conformations for p52 and PSel kB account for favorable entropy?

10) The authors include Bcl3 in a set of binding kinetics experiments and get similar relative rates as with p52 alone. However, the idea that Bcl3 could have an influence on site-specific transcriptional activity seems to have been ruled out because Bcl3 doesn't contact the kB site. What about an indirect mechanism? The fact that unmodified Bcl3 prevents p52 from binding DNA, yet phospo-Bcl3 does not is very suggestive of some level of involvement.

Reviewer #1 (Recommendations for the authors):

Meshcheryakov et al. present a compelling story showing that upon binding to DNA, an NF-κB homodimer induces distinct conformations in DNA, regardless of the DNA sequence or their level of transcriptional activity. To understand how the protein achieves transcriptional selectivity for distinct sequences, authors use simulations, binding assays, and kinetic studies. They observe a correlation between kinetics and transcriptional activation, showing that binding to active DNA sequences is driven by entropy and occurs without a large deformation of the DNA. Conversely, binding to DNA sequences with lower transcriptional activity requires more significant alteration of DNA conformation and is largely enthalpic with slower kinetics. These studies uncover an important role of binding kinetics in DNA selectivity by transcription factors that should motivate many future studies to further explore and elucidate these mechanisms.

I found this study very compelling and believe that this manuscript represents an important contribution to our understanding of DNA selectivity in TFs. To my knowledge, the role of binding kinetics has not been appreciated or investigated thoroughly and needs to be considered more, which will be inspired by this work. While I do not have any concerns that should prevent or even delay publication, I have provided a few suggestions below that I believe may enhance readability and should only improve an already strong piece of work.

Lines 76-80: authors should consider just writing out the kB consensus sequences (5'-GGGNNNNNCC-3') and explaining the numbering separately, as the current form is hard to read/parse.

Authors should include the full name of NF-κB, I don't believe I found any mention of it throughout the manuscript.

I think a few things could help to enhance the introduction section. As written, the final paragraph of this section only vaguely hints at but does not highlight the major results of the paper. Including the full sequence of psel kB, mentioning which promoters are active vs inactive, and how the MD and biochemical data help to distinguish between these where structures do not would help to strengthen this section. Basically, more specific details of the results in this section could be very nice!

Related, it could be helpful for the authors to write out the full sequences of the mutant A/T and -1/+1 swap in both introduction and again in results. Yes, readers can infer what each permutation means, but it would be nice to have it explicitly written out to avoid this step.

Line 169 is the first mention of LPS. Please define this.

This is a minor point, but could authors color figure 2D by domain? Even a lighter/darker version of the selected colors would help readers to more easily decipher the interdomain interface and make the figure clearer.

A set of interactions is described in lines 262-266 which are not shown in any figure. It would be nice to either include the figure reference (if there is one) or create a figure. E.g. I could not find Cys57 on any of the figures.

Please clarify lines 289-294. When you say swapped, which DNAs are you referring to explicitly? This part is tricky from the reader's perspective because technically, the A-T swap and control are swapped relative to the others.

Lines 128-130: referring to work by Leung et al. It is not clear what this reference means when it says 'binding of RelA homodimer to A- and T- centric DNAs are different'. Different from what? From binding to GC-centric DNA, or different from what p52 homodimer does?

Can authors specify what atoms are used in the measurement of minor groove width? This is not clear since they show ~ 2 widths per basepair in the Figure 1 table.

lines 245-251: this part got tricky to read because of the back and forth between figures 2 and 3. If there is any way the authors can move Figure 2D to be part of Figure 3, it would help to streamline the reading of this section.

The only part of this study (for me) that seems slightly disconnected from the rest is the crystallographic data from the longer protein and DNA constructs which showed a different organization of the complex. In particular, I am skeptical of the use of the phrase 'correlation between p52 protein length and conformation of kB DNA'. Correlation is a less than ideal word choice because it implies a quantifiable relationship. It is not clear how a crystal structure conformation could be correlated with anything else unless the authors were highlighting a very specific structural feature within either the protein or DNA that changes proportionally with the length. Could authors rephrase all mentions of correlation here to say something like 'observed influence of p52 length on the conformation of kB'?

Lines 418-419: authors make mention of the most populated conformation of the free DNA, but other than examining minor groove widths, the authors have not performed an actual conformational analysis. Authors could perhaps consider performing an RMSD or RMSF analysis, something that lets us see how the structure changes over the simulation or even the bases of DNA that are most flexible or stable over time. Such an analysis would lend support to even the broader claims throughout this paper that the DNA conformation is differentially influenced by protein binding.

Reviewer #2 (Recommendations for the authors):

The overall conclusions of this work are: (i) p52 homodimers are able to recognize kB sites with a G at the central position by naturally fitting the wider minor groove found in those sites, (ii) binding affinity of p52 to kB sites is poorly correlated with transcriptional activity but the entropic contribution to binding is correlated, (iii) higher on and off rates for p52/kB binding are associated with more transcriptionally active kB sites. The results are very interesting and address important questions that underlie mechanisms of transcriptional regulation.

The authors provide data to support their conclusions, but in some cases, the data and interpretations are not entirely convincing. The structural data support a wider minor groove for the PSel kB site, but it is not clear how p52 recognizes this difference. The loss of cross-strand hydrogen bonds near the center of the site is noted, but the addition of a canonical Arg-Gua interaction is shown but not discussed. A higher level of asymmetry in the dimer-DNA interface is described, but it is not clear how this relates to the central 3 bp in question.

The colorimetric studies are fascinating. A range of binding enthalpies from -5 to -15 kcal/mol are reported for the four kB sites studied and a range of -5 to +5 kcal/mol for the enthalpic contribution to binding (TΔS). The more favorable entropic free energy of binding is associated with kB sites that are more transcriptionally active. It is unclear what the source of these wide variations might be; the authors do not attempt to explain the results in light of the structural models.

Binding kinetic studies are also revealing and interesting. The more transcriptionally active kB sites (e.g., PSel kB) have faster on-rates and faster off-rates. The least active kB sites (e.g., MHC) have lower on-rates and lower off-rates. The authors suggest that increased resident time of p52/Bcl3 on the promoter is associated with lower transcriptional activity; a possible reason is that longer times allow the recruitment of co-repressors. The binding affinities derived from BLI experiments are more than 50-fold lower than those measured by ITC. Assuming that the ITC values are correct, this raises questions about what aspects of the kinetic measurements are valid and which might not be.

Overall, I think this is an interesting and important study, but the connections between the structural studies and the binding experiments need to be strengthened and there are several questions/issues about the individual experiments and interpretations that need to be resolved.

I found the paper a little hard to read, partly due to writing errors and the omission of critical data. Below is a list of specific concerns, major things first, then small things.

Table 1. I cannot find this table in the composite pdf (text + Figures+ supplemental). I assume this table contains crystallographic data/results, making it impossible to evaluate the quality of the structures. I have no idea what the resolution even is for any of the structures.

Structural data. The data shown in Figure 1 indicate a wider minor groove for p52 homodimers with C-terminal extensions bound to long (18bp) kB sites (PSel, PSelG->A, pSelAT) and a narrower minor groove for minimal p52 homodimers bound to 13 bp kB sites (pSelAT, MHC). The authors suggest that the protein length may be important, but they do not discuss that the DNA length may also influence the structures. The same kB sequence (PSelAT) appears in both compressed and expanded minor groove forms. Analysis of the structures indicates that contacts (Ser61-A6,A7) are made outside of the 11-bp kB site in the 18-bp structures and would not be present in the 13-bp structures. So, the conclusion that p52 bound to PSel kB has a widened minor groove relative to other complexes may be correct, but it is far from solid given the data that is presented.

What is different about p52-PSel kB contacts compared to kB sites with a central A/T (like MHC)? This is a major question since the overall goal is to understand how a single bp can make such a large difference in the transcriptional outcome, but the answer is not clear to me. Loss of a cross-strand interaction is discussed, but a new Arg-Gua interaction is gained and that is not discussed. There is a lot of detail about the asymmetric nature of the complex, but most of that appears to be common to all kB sites.

Calorimetry. Adding an error estimate for ΔS would allow readers to evaluate whether the differences are significant. There is an amazing 3-fold variation in ΔH, from -5 kcal/mol to -15 kcal/mol. Equally amazing is that the entropic contribution TΔS also varies widely, from +5 kcal/mol to -5 kcal/mol. I found it surprising that there was no attempt to relate these findings to the known structures. Do the number of polar interactions differ? Solvent accessible surface? Amount of ordered solvent?

Kinetics. The Kd values differ by > 50-fold from the calorimetry results. This discrepancy needs to be addressed. Can the on and off-rates be trusted in these experiments? Even if they are relative rates, one might think that their ratios (K) would still be relevant.

Discussion.

1. None of the binding events are 'entropy driven'. The most favorable (PSel kB) is still 50:50 enthalpy:entropy.

2. Why would binding with native conformations for p52 and PSel kB account for favorable entropy?

3. The authors include Bcl3 in a set of binding kinetics experiments and get similar relative rates as with p52 alone. However, the idea that Bcl3 could have an influence on site-specific transcriptional activity seems to have been ruled out because Bcl3 doesn't contact the kB site. What about an indirect mechanism? The fact that unmodified Bcl3 prevents p52 from binding DNA, yet phospo-Bcl3 does not is very suggestive of some level of involvement.

Reviewer #3 (Recommendations for the authors):

Understanding how transcriptional activity is regulated is a very important topic. The authors provide a detailed structural and biophysical characterization of several complexes of the p52 homodimer of NF kB and different DNA binding sites. The focus is on the central base pair as well as the flanking base pairs on both sides. By x-ray crystallography the authors show that the minor grove of the central base pairs is widened with the G:C base pairs compared to the A:T base pairs. Using MD simulations of the DNA the authors show that DNA molecules can adopt different conformations and the binding of the p52 homodimer induces the least conformational changes in the naturally occurring sequence. The authors go further and correlate these conformational changes with binding affinity and with kinetic kon and koff measurements. Overall, there is little correlation between affinity and transcriptional activity. The only correlation they observe is between fast on and off kinetics and higher transcriptional activity. This result is surprising and does not immediately provide a mechanistic interpretation of how high transcriptional activity is achieved. The authors try to provide an explanation via binding of co-repressors but the fundamental biophysical investigations for such a model are missing at the moment.

1) I have a hard time understanding how the raw ITC data are converted into the integrated data shown below the raw data. In principle, the dilution heat has to be subtracted followed by the integration of the peak. In the graph with the integrated data, the first 7 points in A have the same value (as is expected for such a strong binding). But the peaks in the raw data diagram vary substantially.

2) How was the DNA prepared for the ITC measurements? In two of the four diagrams showing the raw data substantial dilution heat of the DNA is measured. Since the DNA sequence is very similar in all cases, a similar dilution heat is expected. Is there any impurity in two of the four preparations (that would also influence the overall results)?

3) How often were the ITC data repeated? Also, the sample contained 1 mM DTT which often provides problems in ITC measurements. TCEP would have been a better choice.

4) The kD values determined with ITC and BLI differ significantly by two orders of magnitude. This needs to be explained.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting the paper entitled "Structures of NF-κB p52 homodimer-DNA complexes rationalize binding mechanisms and transcription activation" for further consideration by eLife. Your revised article has been evaluated by a Senior Editor and a Reviewing Editor. We are sorry to say that we have decided that this submission will not be considered further for publication by eLife at the moment.

We had a long discussion on your paper among the editors and reviewers involved. Everyone thinks that this is a very important topic that in principle should be further investigated and published. However, in addition to not completely satisfactory answers to some technical questions (the question of the difference in dilution heat between almost identical DNA oligos and the differences between ITC and BLI measurements are still not resolved) the main question of this paper remains unanswered. While the title of your manuscript "Structures of NF-κB p52 homodimer-DNA complexes rationalize binding mechanisms and transcription activation" suggests that the manuscript explains the functional differences between the different NF-κB oligo complexes (which would indeed be a tremendous advance in our understanding of transcriptional regulation in general as well as by NF-κB in particular) , the agreement among the reviewers and editors was that at the current stage the manuscript describes some interesting observations but without offering a convincing explanation. In your rebuttal letter you state that "We admit that even with all these structures, we still don't fully understand how a single bp change makes so much difference. We believe that a fundamental difference is in the alteration of DNA dynamics. However, rather than giving a speculative answer, we would like to wait for the results of more experiments, such as longer timescale MD simulations of these protein-DNA complexes". This is a very honest statement -- which we appreciate but is also confirms that there is no real understanding of the mechanism of this important question. During the discussion with the reviewers it became clear that we are all very enthusiastic about the project but would like to see more data that support a model that explains the observed effects. One possibility would be the addition of the longer timescale MD simulations that you mentioned if they add to the clarification of the model.

All reviewers explicitly stated that they want to be involved in the evaluation of a revised manuscript and support the submission of a manuscript including the MD data.

https://doi.org/10.7554/eLife.86258.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Essential revisions:

1) The resolution obtained for the crystal structures does not allow the identification of individual hydrogen bonds and other details. These limitations should be discussed since these details are important for the structural interpretation.

The resolution issue has been clarified in lines 220-222: It should be noted that all the complex structures are at ~3.0 Å resolution, which places limits on the identification of some detailed interactions, including hydrogen bonds.

2) The data shown in Figure 1 indicate a wider minor groove for p52 homodimers with C-terminal extensions bound to long (18bp) kB sites (PSel, PSelG->A, pSelAT) and a narrower minor groove for minimal p52 homodimers bound to 13 bp kB sites (pSelAT, MHC). The authors suggest that the protein length may be important, but they do not discuss that the DNA length may also influence the structures. The same kB sequence (PSelAT) appears in both compressed and expanded minor groove forms. Analysis of the structures indicates that contacts (Ser61-A6,A7) are made outside of the 11-bp kB site in the 18-bp structures and would not be present in the 13-bp structures. So, the conclusion that p52 bound to PSel kB has a widened minor groove relative to other complexes may be correct, but it is far from solid given the data that is presented.

Various DNA lengths ranging from 12 to 20 bp were used in the co-crystallization, and the details have been added to Supplementary File 1. The long p52 (aa 1-398) protein crystallized with 18 and 20 bp PSel-κB DNAs and diffracted better with the 18 bp DNA. The short p52 (aa 1-327) protein only crystallized with the PSel (mutant A/T-centric) DNA at 13 bp length but not any longer DNAs; it did not crystallize with the PSel (natural G/C-centric) or (-1/+1 swap) DNAs at any length. This has been added in lines 214-217 and lines 254-257. In addition, as mentioned by the reviewer, we also mentioned in the text that “Ser61 also makes direct contact with A at ±6 and ±7 positions; these contacts are not possible for the short p52 (aa 1-327) co-crystallized with 13bp κB DNAs such as MHC and PSel (mutant A/T-centric)-κB (Figure 3—figure supplement 1A-B)” (lines 296-299).

The length and sequence of DNA also influence the structures, and we believe they are corelated with the length of p52 protein (lines 267-268). The direct contacts between the protein and A at ±6 and ±7 base pairs are a direct example of this. We did not emphasize the discussion on the ±6 and ±7 contacts because we do not fully understand the connectivity between the protein and DNA sequence and lengths.

3) The main question of the manuscript is what is different about p52-PSel kB contacts compared to kB sites with a central A/T (like MHC)? The overall goal is to understand how a single bp can make such a large difference in the transcriptional outcome, but the answer is not clear. Loss of a cross-strand interaction is discussed, but a new Arg-Gua interaction is gained and that is not discussed. There is a lot of detail about the asymmetric nature of the complex, but most of that appears to be common to all kB sites. A better definition and discussion of the structural source of the observed selectivity is necessary.

The reviewer is absolutely correct. We ask the same question and present here our best understanding based on the currently available information. We admit that even with all these structures, we still don’t fully understand how a single bp change makes so much difference. We believe that a fundamental difference is in the alteration of DNA dynamics. However, rather than giving a speculative answer, we would like to wait for the results of more experiments, such as longer timescale MD simulations of these protein-DNA complexes. To connect the difference between binding strategies to transcription is even more challenging. Nonetheless, we think continued careful structural and binding analyses will lead to an answer.

The p52 (aa 1-398) Arg52 in the complex with long 18 bp DNAs loses the cross-strand interaction; however, the NH1 and NH2 groups of Arg52 form H-bonds with both the O6 and N7 groups of G at ±3. The same Arg52 in the complex with the short 13 bp PSel (mutant A/T-centric) or the MHC-κB DNA only hydrogen bonds with the O6 group, but not the N7, of G at ±3. The homologous Arg54 in p50 also only contacts the O6 group of G at −3 in the p50:RelA-IFNb-κB DNA complex. We do not know if these contacts are of similar or different strengths. However, these variations in contacts suggest alterations in DNA dynamics (as discussed below in point #9) when the length or sequence changes. This has been added in lines 279-284.

4) It is not obvious how the raw ITC data are converted into the integrated data shown below the raw data. In principle, the dilution heat has to be subtracted followed by the integration of the peak. In the graph with the integrated data, the first 7 points in A have the same value (as is expected for such a strong binding). But the peaks in the raw data diagram vary substantially. That could be due to differences in the width of the peaks, but that should be explained as why it happens and shown. It would also help to have at least one repetition of the experiments for validation purposes.

We thank reviewer’s suggestion. The ITC experiments in Figure 5 have been repeated using a new batch of oligos and are shown in Supplemental Figure 5—figure supplement 1. In the repeated data, the MHC DNA still shows the highest affinity with p52; and the entropy and enthalpy values follow the same trend as the original data even though the exact values vary.

For the data integration, the area under the curve (gray shadow in Author response image 1) corresponds with the total heat generated, from which ΔH is determined and integrated to a dot. Both the depth and width of the first 7 peaks in panel A vary in the upper raw data diagram, resulting in similar integrated peak areas for the bottom panel. The same is true for the other panels of the figure. A detailed explanation has been added to the corresponding figure legend.

Author response image 1

5) Related: How was the DNA prepared for the ITC measurements? In two of the four diagrams showing the raw data substantial dilution heat of the DNA is measured. Since the DNA sequence is very similar in all cases, a similar dilution heat is expected. Is there any impurity in two of the four preparations (that would also influence the overall results)?

The DNA oligos were dissolved in freshly made ITC buffer (same as the p52 protein). An equal amount of top and bottom strands of the oligos were mixed, followed by heating at 95°C for 10 minutes and slowly cooling down to room temperature for annealing. This has been added to the Materials and methods section for ITC (lines 626-629). The mass-spec data on DNA purity (submitted separately as Related Manuscript Files) shows that all DNA strands are highly pure, suggesting impurity is possibly not the source of the observed differences in dilution heats. Moreover, in the repeat experiments, the same two DNAs (both G/C centric, panels A and C, while the other two are A/T centric) showed larger dilution heats similar to the original experiments, even though they were from a new batch. Therefore, the different dilution heats might be an inherent property of the DNAs.

6) Adding an error estimate for ΔS would allow readers to evaluate whether the differences are significant. There is an amazing 3-fold variation in ΔH, from -5 kcal/mol to -15 kcal/mol. Equally amazing is that the entropic contribution TΔS also varies widely, from +5 kcal/mol to -5 kcal/mol. I found it surprising that there was no attempt to relate these findings to the known structures. Do the number of polar interactions differ? Solvent accessible surface? Amount of ordered solvent?

The error estimates for ΔS are not reported by the software on the ITC instrument. The ITC machine directly generates K (which is 1/Kd) and ΔH with the confidence intervals after each run. Please see Author response image 2 from the ITC machine directly as an example of the data shown in Figure 5A. From the K value (1.27x107 ± 4.75x106 M-1) in the picture, the Kd was calculated by 1/K = [1/(1.27 x107 M-1)]x109 = 78.7 nM.

Author response image 2

We have calculated the surface area, the buried area in the p52 dimer and in the DNA interface, and the H-bonds, which are shown in the table below (and added as the new Supplementary File 2). There is no significant difference among the structures, although it is often not easy to relate thermodynamic or kinetic binding parameters to static structures which do not explain the dynamic path to binding. In addition, the solvent structure in the complexes is not clearly defined due to the limited resolution of the structures.

7) The Kd values determined from the kinetic measurements differ by > 50-fold from the calorimetry results. This discrepancy needs to be addressed. Can the on and off-rates be trusted in these experiments? Even if they are relative rates, one might think that their ratios (K) would still be relevant.

This is often an issue with the binding affinity of a macromolecular complex measured by different methods. We believe that tethering of the DNA to a solid surface in the BLI assays differentially affects its dynamics, an important component in the binding mechanism. However, the advantage of BLI is that it provides the ability to monitor the rates of association and dissociation in real-time through measuring the increase in the optical thickness at the biosensor tip. ITC, on the other hand, uses very high, non-physiological concentrations of the components. The gel-based EMSA is another common method for measuring protein-DNA/RNA affinity, which also shows different values. In that case, the complexes are running through the gel pores in low salt buffers which is far from the native binding conditions. In the end, we think the relative values are more significant than the absolute numbers. The Kd values from BLI assays follow the same trend as those from ITC.

8) None of the binding events are 'entropy driven'. The most favorable (PSel kB) is still 50:50 enthalpy:entropy.

We thank the reviewer for pointing this out. We have revised the writing in the Result section as “binding of p52 to the natural G/C-centric PSel-κB DNA is associated with a large increase in entropy (∆S) and a moderate decrease in enthalpy (∆H). On the other hand, the binding to the MHC and mutant A/T-centric PSel DNAs showed a much larger decrease in enthalpy. These results suggest that the binding of p52:p52 homodimer to the G/C-centric κB DNA is favored more by entropy, whereas the binding to the A/T-centric DNA is driven by enthalpy alone” (lines 391-398). Other places in the discussion have also been revised accordingly.

9) Why would binding with native conformations for p52 and PSel kB account for favorable entropy?

Based on the MD simulations, the most populated conformation of the free G/C-centric PSel DNA is similar to the one observed in the crystal structure of the complex, suggesting this DNA’s conformation does not undergo significant changes upon protein binding. Thus, in the complex between p52:p52 and natural G/C-centric DNA, both the DNA and protein most likely preserve their native states. This could account for the positive entropy and faster kon. However, possibly the protein-DNA contacts in such a complex are sub-optimal, resulting in their faster dissociation. In contrast, p52:p52 complexes with the mutant A/T-centric or −1/+1 swapped DNAs likely involve rigidification of protein-DNA contacts, requiring some structural reorganizations in both molecules, resulting in more enthalpically stable complexes and slower association and dissociation rates. This is included in the Discussion section, lines 484-494.

10) The authors include Bcl3 in a set of binding kinetics experiments and get similar relative rates as with p52 alone. However, the idea that Bcl3 could have an influence on site-specific transcriptional activity seems to have been ruled out because Bcl3 doesn't contact the kB site. What about an indirect mechanism? The fact that unmodified Bcl3 prevents p52 from binding DNA, yet phospo-Bcl3 does not is very suggestive of some level of involvement.

The reviewer is correct in that phosphorylation alters Bcl3’s transcriptional activity. We are currently performing experiments to understand how phospho-Bcl3 becomes a transcriptional cofactor by inducing the formation of the ternary complex. This is counterintuitive since phosphorylation of one of the three residues (S446) in the C-terminus, which possibly lies near the negatively charged DNA, is expected to be repulsive. We believe that there is a unique structural component regulating this event where the C-terminus of phospho-Bcl3 adopts a distinct conformation and moves away from the protein-DNA interface. We hope we will be able to clarify in the future how this phosphorylation-dependent conformational variation dictates Bcl3’s transcriptional activity.

[Editors’ note: what follows is the authors’ response to the second round of review.]

We had a long discussion on your paper among the editors and reviewers involved. Everyone thinks that this is a very important topic that in principle should be further investigated and published. However, in addition to not completely satisfactory answers to some technical questions (the question of the difference in dilution heat between almost identical DNA oligos and the differences between ITC and BLI measurements are still not resolved) the main question of this paper remains unanswered. While the title of your manuscript "Structures of NF-κB p52 homodimer-DNA complexes rationalize binding mechanisms and transcription activation" suggests that the manuscript explains the functional differences between the different NF-κB oligo complexes (which would indeed be a tremendous advance in our understanding of transcriptional regulation in general as well as by NF-κB in particular) , the agreement among the reviewers and editors was that at the current stage the manuscript describes some interesting observations but without offering a convincing explanation. In your rebuttal letter you state that "We admit that even with all these structures, we still don't fully understand how a single bp change makes so much difference. We believe that a fundamental difference is in the alteration of DNA dynamics. However, rather than giving a speculative answer, we would like to wait for the results of more experiments, such as longer timescale MD simulations of these protein-DNA complexes". This is a very honest statement -- which we appreciate but is also confirms that there is no real understanding of the mechanism of this important question. During the discussion with the reviewers it became clear that we are all very enthusiastic about the project but would like to see more data that support a model that explains the observed effects. One possibility would be the addition of the longer timescale MD simulations that you mentioned if they add to the clarification of the model.

We deeply appreciate that all reviewers found our project to be interesting and important to study. Based on reviewers’ suggestions, we have now further revised the manuscript. We have carried out microsecond MD simulations of the three (p52:p52)-DNA complexes, including the natural G/C-centric, mutant A/T-centric and −1/+1 swap PSel-κB DNAs. The complex of p52:p52 homodimer with the more transcriptionally active natural G/C-centric DNA maintains very similar conformations under simulations as seen in the crystal structure. In contrast, induced by their narrowed minor groove geometries, the p52 protein closes on the DNAs in the −1/+1 swap complex and to a lesser extent in the mutant A/T-centric complex, which is likely unfavored by the p52:p52 homodimer conformation. These structural changes led to the cross-strand contacts by Lys144 in the −1/+1 swap and mutant A/T-centric DNAs but not in the natural G/C-centric PSel DNA. Combination of MD simulations and structural studies supports the model as PSel (mutant A/T-centric) and (−1/+1 swap) DNAs undergo conformational changes from free to bound states. The relationship between binding kinetics and conformations of the complexes is further supported by the mutational study of this Lys144 residue. These results have been added to a new section “Differential minor groove geometries correlate with differential DNA binding kinetics” (lines 418-473), with new Figures 7-8, and Figure 7—figure supplement 1-2, Figure 8—figure supplement 1.

We believe, with the addition of the new data, our work highlights the importance of DNA sequence-dependent dynamics in protein-DNA recognitions.

https://doi.org/10.7554/eLife.86258.sa2

Article and author information

Author details

  1. Wenfei Pan

    Faculty of Health Sciences, University of Macau, Taipa, China
    Contribution
    Data curation, Formal analysis, Methodology
    Contributed equally with
    Vladimir A Meshcheryakov and Tianjie Li
    Competing interests
    No competing interests declared
  2. Vladimir A Meshcheryakov

    Faculty of Health Sciences, University of Macau, Taipa, China
    Present address
    Molecular Cryo-Electron Microscopy Unit, Okinawa Institute of Science and Technology Graduated University, Okinawa, Japan
    Contribution
    Data curation, Formal analysis
    Contributed equally with
    Wenfei Pan and Tianjie Li
    Competing interests
    No competing interests declared
  3. Tianjie Li

    Department of Physics, Chinese University of Hong Kong, Shatin, Hong Kong
    Contribution
    Data curation, Formal analysis, Writing - original draft, Writing - review and editing
    Contributed equally with
    Wenfei Pan and Vladimir A Meshcheryakov
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4734-1577
  4. Yi Wang

    Department of Physics, Chinese University of Hong Kong, Shatin, Hong Kong
    Contribution
    Funding acquisition, Writing - review and editing
    Competing interests
    No competing interests declared
  5. Gourisankar Ghosh

    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, United States
    Contribution
    Conceptualization, Supervision, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6311-7351
  6. Vivien Ya-Fan Wang

    1. Faculty of Health Sciences, University of Macau, Taipa, China
    2. MoE Frontiers Science Center for Precision Oncology, University of Macau, Taipa, Macao
    3. Cancer Centre, Faculty of Health Sciences, University of Macau, Taipa, China
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    vivienwang@um.edu.mo
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1984-2713

Funding

Science and Technology Development Fund (0104/2019/A2)

  • Vivien Ya-Fan Wang

University of Macau (Multi Year Research Grant MYRG2018-00093-FHS)

  • Vivien Ya-Fan Wang

Hong Kong Research Grant Council Collaborative Research Fund (C6021-19EF)

  • Yi Wang

Chinese University of Hong Kong

  • Tianjie Li
  • Yi Wang

National Institutes of Health

  • Gourisankar Ghosh

Science and Technology Development Fund (0089/2022/AFJ)

  • Vivien Ya-Fan Wang

Macao SAR (FDCT) (0104/2019/A2)

  • Vivien Ya-Fan Wang

Macao SAR (FDCT) (0089/2022/AFJ)

  • Vivien Ya-Fan Wang

National Institutes of Health (GM085490)

  • Gourisankar Ghosh

National Institutes of Health (CA142642)

  • Gourisankar Ghosh

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors thank the Proteomics, Metabolomics and Drug Development (PMDD) Core and Prof. Qi Zhao at Faculty of Health Sciences for providing the ITC machine for the thermodynamic assays and the BLI machine for the binding assays, respectively. The authors thank the staffs from BL19U1 beamline of National Facility for Protein Science in Shanghai (NFPS) at Shanghai Synchrotron Radiation Facility, for assistance during data collection. The authors thank Prof. Liang Tong at Columbia University for critical discussion of the manuscript. This work was supported by the Science and Technology Development Fund, Macao SAR (FDCT) (project 0104/2019 /A2 and 0089/2022/AFJ to VY-FW); the Multi-Year Research Grant from University of Macau (MYRG2018-00093-FHS to VY-FW); and the computing resources of the X-GPU cluster supported by the Hong Kong Research Grant Council Collaborative Research Fund (C6021-19EF to YW). TL and YW were supported by direct grants from the Chinese University of Hong Kong. GG were supported by the National Institutes of Health (NIH) (GM085490 and CA142642 to GG).

Senior and Reviewing Editor

  1. Volker Dötsch, Goethe University, Germany

Publication history

  1. Preprint posted: May 3, 2022 (view preprint)
  2. Received: January 18, 2023
  3. Accepted: February 7, 2023
  4. Accepted Manuscript published: February 13, 2023 (version 1)
  5. Version of Record published: March 7, 2023 (version 2)

Copyright

© 2023, Pan, Meshcheryakov, Li et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Wenfei Pan
  2. Vladimir A Meshcheryakov
  3. Tianjie Li
  4. Yi Wang
  5. Gourisankar Ghosh
  6. Vivien Ya-Fan Wang
(2023)
Structures of NF-κB p52 homodimer-DNA complexes rationalize binding mechanisms and transcription activation
eLife 12:e86258.
https://doi.org/10.7554/eLife.86258

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