Structural insights into SETD3-mediated histidine methylation on β-actin
- University of Science and Technology of China, China
- University of Warsaw, Poland
- University of Toronto, Canada
Figures
Figure 1 with 8 supplements
The SETD3 core region specifically recognizes a fragment of β-actin containing His73.
(A) Domain architecture of full-length human SETD3 (aa 1–594) and the sequence of the β-actin peptide (66–88), with His73 of β-actin highlighted. (B) Representative ITC binding curves for the …
Figure 1—figure supplement 1
Mass Spectrometry data showing the activity of SETD3 toward different peptides.
The core region of SETD3 (aa 2–502) methylates (A) the β-actin peptide (aa 66–88), but not (B) H3K4 (aa 1–23), (C) H3K36 (aa 25–47), or (D) β-actin H73A (aa 66–88). (E) The activity of SETD3 mutants …
Figure 1—figure supplement 2
Sequence alignment of human SETD3 with its orthologs or other SET domain proteins (SETD6 and LSMT).
(A) Sequence alignment of human SETD3 (Hs, NP_115609.2), mouse SETD3 (Ms, NP_082538.2), Xenopus SETD3 (Xs, NP_001016577), zebrafish SETD3 (Zb, NP_956348.1) and Drosophila SETD3 (Dr, XP_017048262.1). …
Figure 1—figure supplement 3
Topology of SETD3 (aa 2–502), with secondary structures marked and colored as in Figure 1—figure supplement 2A.
https://doi.org/10.7554/eLife.43676.005
Figure 1—figure supplement 4
Superposition of SETD3 molecules.
(A) Superposition of four SETD3 molecules bound to methylated β-actin in the same asymmetric unit, with the four molecules shown in red, green, blue and yellow ribbons. His73me of β-actin and AdoHcy …
Figure 1—figure supplement 5
The 2|Fo|–|Fc| σ-weighted maps of peptide and AdoHcy.
The 2|Fo|–|Fc| σ-weighted maps of (A) methylated β-actin peptide and (B) unmodified β-actin peptide in the presence of AdoHcy are contoured at 1.2 σ (blue cage). The peptides are shown in yellow …
Figure 1—figure supplement 6
Comparison of AdoHcy's mode of binding to SETD3, LSMT or SETD6.
(A) A detailed depiction of intermolecular interactions between SETD3 and AdoHcy, with AdoHcy shown in cyan sticks, and the AdoHcy binding residues of SETD3 shown in sticks and colored as in Figure …
Figure 1—figure supplement 7
Superposition of AdoMet-bound SETD3 (PDB id: 3SMT) and AdoHcy-bound SETD3-actin.
In the structure of AdoMet-bound SETD3, the protein is shown in a gray ribbon and AdoMet is shown in gray sticks. In the structure of AdeHcy-bound SETD3 in the presence of actin, the SETD3 protein …
Figure 1—figure supplement 8
The overall structure of SETD3 with methylated β-actin peptide in the presence of AdoHcy.
SETD3 is colored and labeled as in Figure 1C. AdoHcy and His73me are shown in sticks.
Figure 2 with 3 supplements
Molecular mechanism of substrate recognition and histidine methylation by SETD3.
(A) The β-actin peptide binds into a long groove on the surface of the SETD3 N-lobe region (right), with His73 of β-actin positioned into a hydrophobic pocket (left). His73 and AdoHcy are shown in …
Figure 2—figure supplement 1
Detailed interactions of SETD3 and β-actin peptide(66–88).
The β-actin peptide mainly contacts with the N-terminal lobe of SETD3, while its N-terminal and C-terminal sides contact the SET and iSET domains, respectively. In the top panel, SETD3 is shown in …
Figure 2—figure supplement 2
Interactions between SETD3 and peptide.
Schematic of the detailed interactions (A) between SETD3 and unmodified β-actin and (B) between SETD3 and methylated β-actin. SETD3 residues are colored as in Figure 1A, while the peptide residues …
Figure 2—figure supplement 3
Actin undergoes conformational changes upon binding to SETD3.
β-actin peptide(66–88) (yellow cartoon) is overlaid with the same fragment in the structure of native β-actin (red cartoon) (PDB id: 1HLU). Native β-actin decomposes its local secondary structures …
Figure 3
Effect of pH on SETD3 enzyme activity.
The pH-dependence of human SETD3 was determined with the use of a purified recombinant N-terminal His6-tagged SETD3 protein (aa 2–502). The enzyme preparation (0.045 µg protein) was incubated at …
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Figure 3—source data 1
Radiochemical measurements of SETD3-dependent methylation of either human recombinant β-actin or its mutated form (H73A) in the presence of increasing pH values of the reaction mixture.
- https://doi.org/10.7554/eLife.43676.021
Tables
Table 1
Binding affinities of SETD3 proteins to β-actin peptides.
https://doi.org/10.7554/eLife.43676.011| β-actin peptide | SETD3 (aa 2–502) | Stoichiometric coefficient (N) | Kd (µM) |
|---|---|---|---|
| 66TLKYPIEHGIVTNWDDMEKIWHH88 | Wildtype | 1.0 | 0.17 ± 0.04 |
| 66TLKYPIEAGIVTNWDDMEKIWHH88 (H73A) | Wildtype | 1.3 | 0.45 ± 0.08 |
| 1ARTKQTARKSTGGKAPRKQLATK23 (H3K4) | Wildtype | *N/A | *NB |
| 25ARKSAPATGGVKKPHRYRPGTVA47 (H3K36) | Wildtype | N/A | NB |
| 66TLKYPIEHGIVTNWDDMEKIWHH88 | R215A | 1.2 | 3.6 ± 1.0 |
| 66TLKYPIEHGIVTNWDDMEKIWHH88 | N256A | 0.94 | 2.1 ± 0.4 |
| 66TLKYPIEHGIVTNWDDMEKIWHH88 | N256D | 1.3 | 23 ± 7 |
| 66TLKYPIEHGIVTNWDDMEKIWHH88 | N256Q | 1.1 | 2.5 ± 0.5 |
| 66TLKYPIEHGIVTNWDDMEKIWHH88 | W274A | 0.94 | 0.51 ± 0.10 |
| 66TLKYPIEHGIVTNWDDMEKIWHH88 | Y313F | 0.86 | 3.0 ± 0.1 |
| 66TLKYPIEHGIVTNWDDMEKIWHH88 | R316A | 1.1 | 7.4 ± 0.9 |
| 66TLKAPIEHGIVTNWDDMEKIWHH88 (Y69A) | Wildtype | 1.4 | 3.8 ± 0.8 |
| 66TLKYPAEHGIVTNWDDMEKIWHH88 (I71A) | Wildtype | 1.3 | 16 ± 3 |
| 66TLKYPIEHGIVTNWADMEKIWHH88 (D80A) | Wildtype | 1.3 | 3.5 ± 0.4 |
| 66TLKYPIEHGIVTNWDAMEKIWHH88 (D81A) | Wildtype | 1.3 | 2.0 ± 0.5 |
| 66TLKYPIEHGIVTNWDDAEKIWHH88 (M82A) | Wildtype | 1.2 | 0.89 ± 0.08 |
| 66TLKYPIEHGIVTNWD80 | Wildtype | 1.0 | 2.9 ± 0.7 |
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*N/A: Not applicable. *NB: no binding affinity detectable by ITC.
Dissociation constants (Kds) were derived from a minimum of two experiments (mean ± S.D.). The original binding curves are shown in Supplementary file 1.
Table 2
Data collection and refinement statistics.
https://doi.org/10.7554/eLife.43676.012| SETD3–β-actin | SETD3-methylated β-actin | |
|---|---|---|
| Protein Data Bank ID | 6ICV | 6ICT |
| Data collection | ||
| Radiation wavelength (Å) | 0.9789 | 0.9792 |
| Space group | P 1 21 1 | P 1 21 1 |
| Cell dimensions | ||
| a, b, c (Å) | 60.19, 175.17, 66.50 | 59.98, 176.69, 125.89 |
| α, β, γ (°) | 90, 92.57, 90 | 90, 93.37, 90 |
| Resolution (Å) | 35.00–2.15 (2.23–2.15) | 72.27–1.95 (2.06–1.95) |
| Rmerge | 0.135 (0.609) | 0.169 (0.868) |
| I / σI | 14 (3.4) | 8.1 (2.6) |
| CC1/2 | 0.988 (0.884) | 0.991 (0.827) |
| Completeness (%) | 100(100) | 99.1 (99.7) |
| Redundancy | 6.4 (6.4) | 7.0 (7.0) |
| Refinement | ||
| Resolution (Å) | 35.00–2.15 | 59.87–1.95 |
| No. of reflections (used/free) | 71998/3629 | 187163/9084 |
| Rwork/Rfree | 0.168/0.205 | 0.178/0.214 |
| Number of atoms/B-factor (Å2) | 8493/30.7 | 16956/33.7 |
| Protein | 7574/30.3 | 15147/33.1 |
| Peptide | 276/32.9 | 591/38.4 |
| AdoHcy | 52/19.3 | 104/20.0 |
| Solvent | 591/36.2 | 1114/40.0 |
| RMSD bonds (Å)/angles (°) | 0.007/0.84 | 0.007/0.87 |
| Ramachandran Plot favored/allowed/outliers (%) | 98.76/1.24/0 | 98.98/1.02/0 |
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Values in parentheses are for the highest-resolution shell.
Table 3
Kinetic properties of wildtype SETD3 and its mutants.
Kinetic parameters were determined by using purified recombinant N-terminal His6-tagged SETD3 proteins.
| SETD3 | Substrate | Kinetic parameters | |||
|---|---|---|---|---|---|
| Vmax | KM | kcat | kcat/KM | ||
| nmol min−1 mg−1 | µM | min−1 | min−1 µM−1 | ||
| Wildtype | β-actin | 13.550 ± 0.364 | 0.502 ± 0.041 | 0.809 | 1.612 |
| R75A | 0.037 ± 0.002 | 0.565 ± 0.071 | 0.002 | 0.004 | |
| R215A | 3.553 ± 0.068 | 1.087 ± 0.056 | 0.212 | 0.195 | |
| N256A | 0.512 ± 0.019 | 0.449 ± 0.053 | 0.031 | 0.069 | |
| N256D | 0.973 ± 0.017 | 0.305 ± 0.0196 | 0.058 | 0.190 | |
| N256Q | 10.860 ± 0.172 | 0.784 ± 0.035 | 0.649 | 0.828 | |
| N278A | 0.019 ± 0.000 | 0.047 ± 0.009 | 0.001 | 0.021 | |
| Y313F | 0.367 ± 0.005 | 0.581 ± 0.023 | 0.022 | 0.038 | |
| R316A | 2.903 ± 0.0477 | 1.115 ± 0.049 | 0.173 | 0.155 | |
| Wildtype | AdoMet | 11.260 ± 0.466 | 0.111 ± 0.020 | 0.673 | 6.063 |
| R75A | 0.265 ± 0.072 | 3.601 ± 1.363 | 0.016 | 0.004 | |
| R215A | 3.666 ± 0.078 | 0.165 ± 0.013 | 0.219 | 1.327 | |
| N256A | 1.136 ± 0.038 | 0.686 ± 0.052 | 0.068 | 0.099 | |
| N256D | 0.671 ± 0.021 | 0.165 ± 0.019 | 0.040 | 0.242 | |
| N256Q | 6.368 ± 0.266 | 0.103 ± 0.019 | 0.380 | 3.689 | |
| N278A | 0.018 ± 0.001 | 0.350 ± 0.042 | 0.001 | 0.003 | |
| Y313F | 1.623 ± 0.348 | 6.543 ± 1.736 | 0.097 | 0.015 | |
| R316A | 4.099 ± 0.054 | 0.149 ± 0.008 | 0.244 | 1.638 |
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Determinations for S-adenosyl-L-methionine (AdoMet) were performed with the SETD3 preparations (0.05–5.00 µg protein, 8.38–838 nM), which were incubated for 10 min at 37°C in a reaction mixture containing 5 µM recombinant human β-actin and variable concentrations of [1H+3H] AdoMet (≈330 × 103 cpm). The measurements for β-actin were done following a 10 min incubation of SETD3 in the presence of a 0.8 μM concentration of [1H+3H] AdoMet (80 pmol, 300–700 × 103 cpm). In all experiments, the reaction mixture contained the homogenous recombinant AdoHcy nucleosidase (1.6 µg protein, 600 nM, E. coli) and adenine deaminase (3.9 µg protein, 600 nM, B. subtilis) to prevent S-adenosyl-L-homocysteine (AdoHcy) accumulation. Values are the means of three separate experiments. The S.E. values are also given.
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Table 3—source data 1
Determination of the kinetic parameters of SETD3-catalyzed methylation of actin (for β-actin as the substrate).
- https://doi.org/10.7554/eLife.43676.018
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Table 3—source data 2
Determination of kinetic parameters of SETD3-catalyzed methylation of actin (for AdoMet as the substrate).
- https://doi.org/10.7554/eLife.43676.019
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| ene (E. coli) | AdoHcy nucleosidase | NA | NCBI: NC_000913.3 | |
| Gene (B. subtilis) | adenine deaminase | NA | NCBI: NC_000964.3 | |
| Gene (Homo sapiens) | β-actin | NA | GenBank: NM_001101.4 | |
| Gene (H. sapiens) | SETD3 | NA | NCBI: NM_032233.2 | |
| Recombinant DNA reagent | pCOLD I (plasmid) | Takara Bio | 3361 | |
| Genetic reagent (E. coli) | pCOLD I/β-actin | PMID: 20851184 | Kind gift from M. Tamura, Ehime University | |
| Genetic reagent (E. coli) | pCOLD I/AdoHcy nucleosidase | PMID: 30526847 | Overexpression of the recombinant E. coli AdoHcy nucleosidase in E. coli BL21 (DE3) | |
| Genetic reagent (E. coli) | pCOLD I/adenine deaminase | PMID: 30526847 | Overexpression of the recombinant B. subtilis adenine deaminase in E. coli BL21 (DE3) | |
| Strain, strain background (B. subtilis) | B. subtilis | Sigma-Aldrich | ATCC:6633 | |
| Strain, strain background (E. coli) | E. coli BL21 (DE3) | Agilent Technologies | 200131 | |
| Chemical compound, drug | S-[methyl-3H]adenosyl-L-methionine; [3H]SAM | PerkinElmer | NET155V250UC; NET155V001MC | |
| Software, algorithm | GraphPad Prism | GraphPad Software | RRID:SCR_002798 | |
| pET28-MHL | Vector | Addgene | 26096 (Genbank accession number: EF456735) | Expression of SETD3 and its mutants |
| Origin 6.1, Origin 7.0 | Software | OriginLab | http://www.originlab.com/ | For ITC curve fitting and calculation of Kd values |
| HKL2000 | Software | PMID: 27754618 | http://www.hkl-xray.com/ | Processing the crystal structure data |
| Phaser | Software | PMID: 19461840 | http://www.phaser.cimr.cam.ac.uk/ | Molecular replacement |
| Phenix | Software | PMID: 20124702 | http://www.phenix-online.org/ | Structure refinement |
| Coot | Software | PMID: 15572765 | http://www2.mrc-lmb.cam.ac.uk/Personal/pemsley/coot/ | Structure refinement |
| Pymol | Software | DeLano Scientific LLC | http://www.pymol.org/ | Making structure figures |
| Molprobity | Software | PMID: 20057044 | http://molprobity.biochem.duke.edu/ | Structure validation |
Additional files
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Supplementary file 1
ITC binding curves for the binding measurements reported in Table 1.
- https://doi.org/10.7554/eLife.43676.022
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Transparent reporting form
- https://doi.org/10.7554/eLife.43676.023
