A structural code for assembly specificity in GID/CTLH-type E3 ligases
- Institute of Structural Biology, Rudolf Virchow Center for Integrative and Translational Bioimaging, Julius-Maximilians-Universität Würzburg, Germany
Figures
Figure 1 with 3 supplements
Shared structural architecture of CTLH-CRA domains.
(A) Domain architecture of the core subunits containing LisH (L), CTLH, and CRA motifs. (B) Crystal structure of the RanBP10-Twa1 complex (3.2 Å, PDB: 9SOC). LisH and CRAC helices are labeled. (C) Schematic representation of the modular CTLH core complex highlighting interchangeable subunits (RanBP9 ↔ RanBP10; muskelin ↔ Wdr26) and variable substrate receptors (Gid4-Armc8, Ypel5, and FAM72A), illustrating the combinatorial nature of subunit arrangements. Gray arrows mark CTLH-CRAN/CTLH-CRA domain heterodimerization interfaces in a composite model of known structures (PDB: 8PJN, 7NSC, 8QBN, 8TTQ, and newly solved 9SOC) fitted into available cryo-EM maps (EMDB: 12542, 12545, 12547, 17713, 17715, 18172, 45088, 45138, and 45186). (D) Schematic overview of CTLH-CRAN/CTLH-CRA domain fusion constructs showing their position and binding specificity within the CTLH core complex. Crystal structures of RanBP9, RanBP10, Twa1, Maea, and muskelin (from RanBP9-muskelin complex) reveal a conserved fold of the CTLH-CRA domain.
Figure 1—figure supplement 1
Conservation of mammalian core CTLH complex subunits.
Overlay of the experimentally determined CTLH-CRA domain structures (PDB: 9SNH, 9SNI, 9SNE, 9SNF, and 9SNV) with full-length AlphaFold predictions (Varadi et al., 2024) of the proteins used in this study (mouse; rat muskelin). Sequence differences between the murine or rat proteins and their human counterparts are shown in black, and variations located within the CTLH-CRA domains are explicitly indicated at the respective positions in the structures. Percent sequence identity to the human proteins is provided. The overlay demonstrates that the truncated and fused CTLH-CRA constructs accurately reflect the structural context of the full-length proteins and highlights the high conservation of these mammalian subunits.
Figure 1—figure supplement 2
Comparison of LisH-CRAC interfaces.
Sequence alignment and logo (top) of LisH motifs based on depicted structures highlight conserved hydrophobic residues, like leucine or phenylalanine (marked with * or circled *). LisH motifs of murine and human Twa1, as well as rat and human muskelin, are identical. Gray shading marks the folding of the two helices, which explains a conserved glycine residue in the loop between them. Another conserved residue near the C-terminus is a glutamate which mediates backbone interactions with the opposing helix. Structural comparison of available LisH-CRAC dimers reveals a conserved helical arrangement. Muskelin was omitted from this comparison because it lacks a CRAC helix, while Lis1 and TBL1 have a CRAC helix despite lacking a CRAN segment. Black lines between the RanBP10 and Twa1 sequences indicate potential hydrogen bonds that may explain why Twa1 preferentially binds RanBP10 rather than forming homodimers.
Figure 1—figure supplement 3
Biochemical characterization of different CTLH-CRA domain complexes.
(A) Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) analyses of co-expressed CTLH-CRA domain complexes of RanBP9-muskelin and RanBP10-muskelin (shortened C-terminus). Measured molar masses match theoretical values (theo.), thus confirming a 1:1 stoichiometry. (B) SEC-MALS measurements of full-length dimeric Twa1, co-expressed with CTLH-CRA domains of Maea or Rmnd5a, reveal comparable 1:1 binding ratios. Twa1 homodimerization (LisH-CRAC-mediated) remains unaffected. Rmnd5a CTLH-CRA domain is soluble when co-expressed with Twa1. (C) Native agarose gel electrophoreses (NAGE) of RanBP10 mutants carrying combinations of Twa1-like substitutions (F543L, Y548F, V556F; L--, -F-, -FF, LF-, L-F, -FF, LFF) illustrate the progressive loss of muskelin binding with increasing substitutions (compare isothermal titration calorimetry [ITC], Figure 2). (D) SEC-MALS analysis of ITC-derived complexes (Figure 5). Addition of excess RanBP9-Twa1 to pre-assembled Twa1_52-muskelin or Twa1_52-Wdr26 complexes results in displacement of monomeric Twa1_52 and formation of muskelin/Wdr26-RanBP9-Twa1 complexes. The oligomeric state of muskelin (tetramer) and of Wdr26 (dimer) remains preserved.
Figure 2 with 2 supplements
Structural determinants of RanBP9/10-muskelin CTLH-CRA specificity.
(A) 2.0 Å crystal structure of the RanBP9-muskelin CTLH-CRA domains (PDB: 9SNV) with close-up view of the interaction site. (B) Sequence alignment of CRA-interface residues highlighting the conserved leucine (marked *). Mouse sequences were used, except for rat muskelin; all are highly similar to the human homologs (Figure 1—figure supplement 1). (C) Molecular details of the RanBP9-muskelin interface overlaid with RanBP10 (PDB: 9SNF) and Twa1 (PDB: 9SOC). (D) Isothermal titration calorimetry (ITC) binding analysis of muskelin with various RanBP10 mutants and the muskelin mutant F686L with RanBP10. (E) Overlay of the RanBP9-muskelin interface with Maea (PDB: 9SNH). (F) Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) analysis of Maea and the double mutant (Y272A, Y275A). (G) 1.4 Å crystal structure of the Maea CTLH-CRA domain (PDB: 9SNH) showing details of the homodimerization interface.
Figure 2—figure supplement 1
Isothermal titration calorimetry (ITC) binding analysis of muskelin and RanBP10.
Representative ITC titrations of CTLH-CRA domains of muskelin and RanBP10. The muskelin F686L mutant was titrated against wild-type (wt) RanBP10, and muskelin wt was titrated against RanBP10 binding mutants (F543L, Y548F, V556F; and combinations thereof: L--, -F-, -FF, LF-, L-F, -FF, LFF), corresponding to Figures 2D and 3B. KD values and molar ratios (n) are shown as mean ± standard error from 3 to 4 replicates (6 for wt). Based on enthalpy and KD values, entropy (ΔS) and Gibbs free energy (ΔG) were calculated, revealing both enthalpic and entropic contributions to the binding reaction. Binding of the RanBP10-Twa1 complex to full-length muskelin was too tight and could not be fitted.
Figure 2—figure supplement 2
Concentration-dependent oligomerization of Maea and RanBP10 Q519G.
(A) Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) molar mass profiles of Maea CTLH-CRA domain across the elution peak illustrate a concentration-dependent oligomerization. The most stable 0.3 ml peak fraction was used for analysis (middle panel). Molar masses were plotted against concentration, derived from the protein amount based on the refractive index (RI) signal in the peak. KD best-fit parameter and 95% confidence interval of the one-site binding model are indicated, representing the dimerization of stable dimers. The theoretical molar mass (theo.) of a dimer was used for normalization. (B) Equivalent analysis of the RanBP10 Q519G mutant. (C) Muskelin-binding-impaired RanBP10 mutants (F543L, Y548F, V556F; and combinations thereof: L--, -F-, -FF, LF-, L-F, -FF, LFF) and wild-type (wt) RanBP10 are stable monomeric CTLH-CRA domains. (D) Structural overlay of CTLH-CRA domain interfaces of wt RanBP9 (similar to wt RanBP10), R10_GLFF, and Maea shows that Q552 (Q519 in RanBP10) restricts Maea binding and dimerization compared to the Q519G mutation.
Figure 3
Rewiring the binding specificity of RanBP10 from muskelin to Maea.
(A) Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), (B) isothermal titration calorimetry (ITC), and (C) native agarose gel electrophoresis (NAGE) binding analysis of CTLH-CRA domains of muskelin and Maea with wild-type RanBP10, R10_G (Q519G), and R10_GLFF (Q519G, F543L, Y548F, V556F) mutants. (D) 1.7 Å crystal structure of the R10_GLFF CTLH-CRA domain (PDB: 9SNG) showing details of the homodimerization interface. Twa1-like mutations are highlighted in yellow. (E) 3.5 Å crystal structure of the R10_GLFF-Maea CTLH-CRA complex (PDB: 9SOH) and overlay (F) with the RanBP9-muskelin CTLH-CRA complex (PDB: 9SNV), illustrating the determinants of binding specificity.
Figure 4 with 3 supplements
Rewiring the binding specificity of Twa1 from Maea to muskelin.
(A) Native agarose gel electrophoresis (NAGE) binding analysis of various Twa1 mutants to Maea and muskelin, accompanied by a depiction of Twa1 sequence changes. The wild-type sequence corresponds to mouse Twa1 (identical to the human ortholog in the interface region) (B) 3.3 Å crystal structure of the Twa1_14-Maea complex (PDB: 9SOI) showing molecular details of the interface, overlaid with muskelin (PDB: 9SNV). (C) Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) analysis of wild-type Twa1, Twa1_14, and Twa1_52 in complex with Maea or muskelin. (D) Isothermal titration calorimetry (ITC) analysis demonstrating that Twa1_14 and Twa1_52 bind muskelin, in contrast to wild-type Twa1.
Figure 4—figure supplement 1
Sequence overview of Twa1 CTLH-CRA domain mutants.
To swap binding specificity and render the murine Twa1 CRA interface more RanBP9/10-like, a total of 52 different Twa1 mutants were purified and tested for binding (see Figure 4—figure supplement 2).
Figure 4—figure supplement 2
Native agarose gel electrophoresis (NAGE) binding analysis of Twa1 CTLH-CRA domain mutants.
Binding of 52 different Twa1 mutants (Figure 4—figure supplement 1) was analyzed at 200 μM against Maea CTLH-CRA (native binding partner) and muskelin CTLH-CRA (acquired RanBP9/10-like binding preference). NAGE served as a screening assay to identify promising candidates for subsequent biophysical validation.
Figure 4—figure supplement 3
Isothermal titration calorimetry (ITC) binding analysis of muskelin and Twa1 mutants.
ITC binding analysis analogous to Figure 2—figure supplement 1, in which muskelin was titrated against Twa1 CTLH-CRA domain mutants (see Figure 4—figure supplement 1). Representative titrations corresponding to Figure 4D are shown. KD values and molar ratios (n) are presented as mean ± standard error from 3 to 4 replicates. Based on calculated entropy (ΔS) and Gibbs free energy (ΔG), the optimization process reduced unfavorable entropic contributions to binding.
Figure 5
Picomolar RanBP9/10 binding to muskelin and Wdr26.
(A) Analytical size exclusion chromatography of RanBP9/10-Twa1 complexes in the presence of muskelin or Wdr26. (B) Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) analysis of co-expressed CTLH-CRA domain complexes Twa1_52-Wdr26 and Twa1_52-muskelin. Theoretical MW is denoted (theo.). (C) Isothermal titration calorimetry (ITC) binding analysis of Twa1_52 to muskelin/Wdr26, followed by competitive titration with RanBP9/10-Twa1 complexes. KD values and stoichiometries (n) are indicated together with the corresponding enthalpic and entropic contributions (below). (D) Cryo-EM maps of the mammalian CTLH complex fitted with available structural data highlight the CTLH-CRA dimerization interface in the context of the full-length assembly. An AlphaFold3 prediction of the RanBP9-Wdr26 CTLH-CRA domain overlaid with the RanBP9-muskelin structure (PDB: 9SNV) illustrates their conserved mode of specificity.
Figure 6 with 4 supplements
CTLH-CRA domain specificity code.
Crystal structures determined in this study of CTLH-CRA domains highlight the specificity of the interaction interfaces and the position of the conserved leucine residue (*) for comparison. Mutational analyses demonstrate that binding specificity can be rewired bidirectionally: the RanBP10 quadruple mutant R10_GLFF (Q519G, F543L, Y548F, and V556F) successfully switched its preference from muskelin to Maea, while the engineered Twa1_52 mutant (A125G, Q126R, Δ127T, L140K, E144K, L147F, F152Y, and F160V) acquired RanBP10-like specificity toward muskelin. Together, these structures and functional swaps define the residue-level code underlying partner recognition within CTLH-CRA interfaces.
Figure 6—figure supplement 1
Overlay of AlphaFold3-predicted CTLH-CRA interfaces across species.
AlphaFold3 predictions of human CTLH-CRA interfaces (Twa1-Rmnd5a, Twa1-Maea, RanBP9-muskelin, RanBP9-Wdr26, RanBP10-muskelin, and RanBP10-Wdr26) were compared with corresponding homologous interfaces from model organisms: Mus musculus, Xenopus laevis, Danio rerio, Caenorhabditis elegans, Drosophila melanogaster, Saccharomyces cerevisiae, and Arabidopsis thaliana (top to bottom). UniProt accession numbers are indicated in gray. Only protein sequences lacking the LisH-CRAC segment were used to avoid a false interface prediction. When Rmnd5a was present, the closely related paralog Rmnd5b was omitted due to their high similarity (human, mouse). In D. rerio, which lacks Rmnd5a, the paralog Rmnd5b was used for prediction. This species also encodes two Twa1 homologs, Gid8a and Gid8b; only Gid8a is shown for clarity. D. rerio further contains a RanBP10 paralog, which X. laevis lacks. For S. cerevisiae, the experimental cryo-EM structure of Gid 1 (RanBP9 homolog) in complex with Gid7 (Wdr26 homolog) was used instead of a prediction for comparison (Figure 6—figure supplement 2). For M. musculus, the prediction was replaced by the experimental RanBP9-muskelin crystal structure.
Figure 6—figure supplement 2
CTLH-CRA interface of the yeast Gid1-Gid7.
(A) Cryo-EM structure of the S. cerevisiae GID complex (PDB: 7NSB), the yeast homolog of the CTLH complex. (B) Structural overlay of murine RanBP9 (from PDB: 9SNV) with its yeast homolog Gid1. (C) Close-up view of the Gid1-Gid7 CTLH-CRA interface. The N-terminus of Gid1 forms an additional contact with the Gid7 interface. The conserved leucine residue (*) is indicated for reference.
Figure 6—figure supplement 3
Non-CTLH complex CTLH-CRA domains.
(A) Structures of CRA interfaces from non-CTLH complex proteins – SMU1, Wdr47, TOPLESS, and TPR2 – reveal a conserved fold extending beyond the CTLH complex. (B) Sequence alignment based on CTLH-CRA domain structures (A), Figure 2B highlights three positional classes of the conserved aromatic residue (dark gray). Overlay of Maea and TRP2 homodimers illustrates that in non-CTLH complex members, the aromatic residue is shifted to the opposite side of the interface, resulting in altered loop geometry. (C) Structures of SMU1, Wdr47, and TRP2 demonstrate that the LisH-CTLH-CRA module can engage additional binding partners beyond the canonical LisH-CRAC and CTLH-CRA interfaces. RED and IAA1 peptides bind to distinct hydrophobic grooves adjacent to the dimerization interface – interaction sites not observed in CTLH complex subunits.
Figure 6—figure supplement 4
Electrostatic surface representation of CTLH-CRA domain structures determined in this study.
Although CTLH-CRA domains share a highly conserved overall fold, their electrostatic surface potential reveals differences beyond the conserved dimerization interface. These variations occur in additional surface grooves that differ in both charge and shape and may represent underexplored, subunit-specific interaction sites. While Twa1 displays an overall negatively charged surface, RanBP9 exhibits a comparatively neutral groove located C-terminally (left panel) to the dimerization interface.
Tables
Table 1
Data collection and refinement statistics of crystal structures from CTLH complex core subunits.
| RanBP9CTLH-CRA | RanBP10CTLH-CRA | R10_GLFFCTLH-CRA | Maea CTLH-CRA | Twa1 CTLH-CRA | Mkln-R9CTLH-CRA | R10_GLFF-MaeaCTLH-CRA | Twa_14-MaeaCTLH-CRA | RanBP10-Twa1 | |
|---|---|---|---|---|---|---|---|---|---|
| PDB ID | 9SNE | 9SNF | 9SNG | 9SNH | 9SNI | 9SNV | 9SOH | 9SOI | 9SOC |
| Wavelength (Å) | 0.9779 | 0.9677 | 0.9763 | 0.9763 | 0.9763 | 0.9763 | 0.9763 | 0.9763 | 0.8731 |
| Space group | P 1 21 1 | P 2 21 21 | P 21 21 2 | P 21 21 21 | P 41 21 2 | P 61 2 2 | F 41 3 2 | P 32 2 1 | P 61 2 2 |
| a, b, c (Å) | 41.277 80.320 45.017 | 55.331 75.500 76.386 | 52.079 68.629 76.453 | 62.075 76.181 108.149 | 118.843 118.843 56.490 | 84.086 84.086 228.282 | 359.825 359.825 359.825 | 142.346 142.346 213.234 | 124.920 124.920 479.645 |
| α, β, γ (°) | 90.00 101.61 90.00 | 90.00 90.00 90.00 | 90.00 90.00 90.00 | 90.00 90.00 90.00 | 90.00 90.00 90.00 | 90.00 90.00 120.00 | 90.00 90.00 90.00 | 90.00 90.00 120.00 | 90.00 90.00 120.00 |
| Resolution limits (Å) | 44.096–1.810 (2.006–1.810) | 44.810–2.069 (2.294–2.069) | 43.042–1.737 (1.870–1.737) | 48.122–1.431 (1.583–1.431) | 46.882–2.113 (2.282–2.113) | 44.919–1.965 (2.150–1.965) | 48.084–3.545 (3.774–3.545) | 48.930–3.307 (3.659–3.307) | 49.307–3.175 (3.788–3.175) |
| Observed reflections | 94,080 (4,377) | 165,652 (9,501) | 313,836 (16,833) | 896,835 (43,613) | 455,867 (22,410) | 948,639 (46,128) | 3,470,467 (207,156) | 353,906 (17,408) | 314,373 (18,872) |
| Unique reflections | 13,616 (681) | 14,606 (859) | 23,475 (1,201) | 67,576 (3,379) | 18,249 (913) | 24,304 (1,215) | 21,897 (1,288) | 17,940 (901) | 11,177 (559) |
| *Rmerge | 0.125 (1.253) | 0.166 (1.797) | 0.153 (1.979) | 0.090 (2.855) | 0.093 (3.060) | 0.188 (6.557) | 0.282 (4.684) | 0.115 (1.987) | 0.367 (3.891) |
| †Rpim | 0.051 (0.528) | 0.051 (0.552) | 0.043 (0.545) | 0.026 (0.822) | 0.019 (0.631) | 0.030 (1.074) | 0.023 (0.368) | 0.026 (0.455) | 0.069 (0.670) |
| CC1/2 | 0.997 (0.445) | 0.995 (0.602) | 0.999 (0.619) | 0.998 (0.429) | 0.999 (0.440) | 0.999 (0.363) | 1.000 (0.331) | 0.999 (0.012) | 0.999 (0.726) |
| ‡<I/σI> | 9.0 (1.5) | 11.5 (1.7) | 13.1 (1.5) | 12.9 (1.0) | 18.3 (1.3) | 16.9 (0.8) | 27.3 (2.3) | 11.1 (1.8) | 9.3 (1.3) |
| Overall completeness spherical/elliptical | 51.8/89.9 (9.9/61.5) | 72.4/91.3 (16.3/57.4) | 81.1/91.7 (21.3/50.7) | 71.1/94.1 (13.8/65.1) | 76.8/93.9 (19.1/61.0) | 69.4/94.9 (15.0/64.2) | 88.5/95.4 (31.2/53.3) | 47.2/93.4 (9.2/82.3) | 88.7/28.9 (49.1/3.6) |
| Multiplicity | 6.9 (6.4) | 11.3 (11.1) | 13.4 (14.0) | 13.3 (12.9) | 25.0 (24.5) | 39.0/38.0 | 158.5 (160.8) | 19.7 (19.3) | 28.1 (33.8) |
| Wilson B-factor (Å2) | 26.91 | 38.32 | 18.39 | 23.28 | 63.11 | 48.24 | 150.24 | 145.19 | 97.08 |
| No. reflections | 13,592 (398) | 14,580 (438) | 23,131 (438) | 67,483 (68) | 18,244 (377) | 24,283 (185) | 21,715 (432) | 17,917 (462) | 10,621 (108) |
| §Rwork/¶Rfree | 0.2005/ 0.2396 | 0.2018/ 0.2494 | 0.2102/ 0.2470 | 0.1989/ 0.2254 | 0.2379/ 0.2645 | 0.2280/ 0.2549 | 0.2824/ 0.2954 | 0.2292/ 0.2630 | 0.2989/ 0.3110 |
| No. of non-hydrogen atoms | 2287 | 2236 | 2341 | 4664 | 2101 | 2229 | 8379 | 4253 | 10,579 |
| Protein | 2242 | 2173 | 2177 | 4278 | 2101 | 2200 | 8379 | 4253 | 10,572 |
| Ligands | 3 | 0 | 0 | 0 | 0 | 5 | 0 | 0 | 7 |
| Solvent | 42 | 63 | 164 | 386 | 0 | 24 | 0 | 0 | 0 |
| Protein residues | 284 | 274 | 275 | 500 | 255 | 272 | 1031 | 509 | 1330 |
| **Ramachandran statistics: favored/allowed (%) | 97.12/2.88 | 99.62/0.38 | 99.63/0.37 | 99.19/0.81 | 98.41/1.59 | 96.59/3.03 | 95.01/4.09 | 96.61/2.14 | 96.74/3.11 |
| Clashscore | 1.55 | 0.69 | 1.84 | 0.94 | 1.20 | 4.33 | 11.36 | 11.12 | 7.43 |
| Overall B-factor (Å2) | 36.15 | 45.76 | 25.40 | 31.92 | 72.62 | 64.16 | 179.72 | 156.98 | 98.05 |
| RMS deviations in | |||||||||
| Bonds (Å) | 0.003 | 0.002 | 0.004 | 0.005 | 0.002 | 0.003 | 0.004 | 0.003 | 0.005 |
| Angles (°) | 0.51 | 0.47 | 0.64 | 0.70 | 0.47 | 0.56 | 0.82 | 0.67 | 0.89 |
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Numbers in parentheses refer to the respective highest resolution data shell in the dataset.
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*
Rsym = ΣhklΣi | Ii –<I > | / ΣhklΣiIi where Ii is the ith measurement and <I > is the weighted mean of all measurements of I.
-
†
Rpim = Σhkl1/(N–1) ½ Σi| Ii(hkl) – I (hkl) | / ΣhklΣiI(hkl), where N is the redundancy of the data and I (hkl) the average intensity.
-
‡
<I / σI>indicates the average of the intensity divided by its standard deviation.
-
§
Rwork = Σhkl ||Fo| – |Fc|| / Σhkl|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively.
-
¶
Rfree same as R for 5% of the data randomly omitted from the refinement. The number of reflections includes the Rfree subset.
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**
Ramachandran statistics were calculated using MolProbity in Phenix.
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A structural code for assembly specificity in GID/CTLH-type E3 ligases
eLife 15:RP110152.
https://doi.org/10.7554/eLife.110152.3
