Two Glu/Asp residues cooperatively mediate an early step of ATP hydrolysis in GHKL ATPases MutL and GyrB
- Department of Biochemistry, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Japan
- Department of Food Science and Nutrition, Faculty of Human Life and Environment, Nara Women’s University, Kitauoyanishi-machi, Japan
- Department of Chemistry, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Japan
Figures
Figure 1 with 1 supplement
Effects of mutations on the overall structure and the ATPase activity of the Aquifex aeolicus MutL (aqMutL) NTD.
(A) Crystal structure of the aqMutL N-terminal domain (NTD) in complex with the non-hydrolyzable ATP analog AMPPCP (6LZJ). The boxed region indicates the ATP-binding site and is shown enlarged on the right. AMPPCP and the side chains of Glu29 and Glu32 are shown as stick models. Oxygen atoms are shown in red, nitrogen atoms in blue, and phosphorus atoms in orange. The magnesium ion and the water molecule putatively acting as the nucleophile are depicted as green and red spheres, respectively. Hydrogen bonds are indicated by dashed lines, with distances shown. The blue mesh represents an omit Fo–Fc electron density map contoured at 3σ for AMPPCP, the magnesium ion, and the water molecule. (B) Sequence alignment of representative GHKL ATPases. The Bergerat ATP-binding fold is underlined, and the conserved acidic residues corresponding to aqMutL Glu29 and Glu32 are marked with asterisks. Amino acid sequence alignment was performed by CLUSTAL W program (Thompson et al., 1994) and visualized by ESPript3 (Gouet et al., 2003). (C) Far-UV CD spectra of the wild-type and mutant forms of the aqMutL NTD were collected and averaged over 10 consecutive scans. (D, E) ATPase activity of the aqMutL NTD was measured under steady-state conditions using a colorimetric assay that detects the release of inorganic phosphate. Apparent rate constants (kapp) were calculated from the measured phosphate concentrations and plotted as a function of ATP concentration. Kinetic parameters were determined by fitting Michaelis–Menten equation to the data. Each data point represents the mean of three independent experiments, with error bars indicating standard deviation. Theoretical curves calculated from the fitted kinetic parameters are overlaid. Data for 0–1 mM substrate concentrations are shown to compare the wild-type form with mutant forms (D). Activities for higher substrate concentrations were measured for some of the mutant forms (E).
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Figure 1—source data 1
This source data file contains the circular dichroism (CD) spectra of wild-type and mutant A. aeolicus MutL NTDs shown in Figure 1C.
- https://cdn.elifesciences.org/articles/111443/elife-111443-fig1-data1-v1.xlsx
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Figure 1—source data 2
This source data file contains the ATPase assay data showing apparent turnover rates (kapp) as a function of ATP concentration for wild-type and mutant A. aeolicus MutL NTDs shown in Figure 1D and E.
- https://cdn.elifesciences.org/articles/111443/elife-111443-fig1-data2-v1.xlsx
Figure 1—figure supplement 1
The ATP-binding ability of the Aquifex aeolicus MutL (aqMutL) N-terminal domains (NTDs) (A) and Aquifex aeolicus GyrB (aqGyrB) NTDs (B).
Binding affinities were quantified by equilibrium dialysis. For each protein concentration, the concentration of unbound AMPPNP was determined from the absorbance at 260 nm of the buffer chamber solution after equilibrium had been reached (see Materials and methods). The concentration of bound AMPPNP was calculated by subtracting the concentration of unbound AMPPNP from the total AMPPNP concentration and was plotted against the protein concentration.
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Figure 1—figure supplement 1—source data 1
This source data file contains equilibrium dialysis measurements of AMPPNP binding to wild-type and mutant A. aeolicus MutL and GyrB NTDs shown in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/111443/elife-111443-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 2 supplements
Crystal structure of the Aquifex aeolicus GyrB (aqGyrB) N-terminal domain (NTD) and effects of mutations on the overall structure and the ATPase activity of the aqGyrB NTD.
(A) Structure of the AMPPNP-bound dimer of the wild-type aqGyrB NTD. The boxed region highlights the ATP-binding site and is enlarged in the left panel. The same region is further enlarged and rotated 60° to provide an alternative view of the active site geometry. AMPPNP and the side chains of Glu48 and Asp51 are shown as stick models. Oxygen atoms are shown in red, nitrogen atoms in blue, and phosphorus atoms in orange. The magnesium ion and the water molecule presumed to act as the nucleophile are depicted as green and red spheres, respectively. Hydrogen bonds are indicated by dashed lines, with distances shown. The blue mesh represents an omit Fo–Fc electron density map contoured at 3σ for AMPPNP, the magnesium ion, and the water molecule. (B) Far-UV CD spectra of the wild-type and mutant forms of the aqGyrB NTD were recorded and averaged over 10 accumulations. (C) ATPase activity of the aqGyrB NTD was measured under steady-state conditions as described in Figure 1. The kapp values were plotted as a function of ATP concentration. Michaelis–Menten equation was fitted to the data. Data points represent the mean ± SD from three independent experiments.
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Figure 2—source data 1
This source data file contains the circular dichroism (CD) spectra of wild-type and mutant A. aeolicus GyrB NTDs shown in Figure 2B.
- https://cdn.elifesciences.org/articles/111443/elife-111443-fig2-data1-v1.xlsx
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Figure 2—source data 2
This source data file contains the ATPase assay data showing apparent turnover rates (kapp) as a function of ATP concentration for wild-type and mutant A. aeolicus GyrB NTDs shown in Figure 2C.
- https://cdn.elifesciences.org/articles/111443/elife-111443-fig2-data2-v1.xlsx
Figure 2—figure supplement 1
Electron density maps around the catalytic acidic residues in the wild-type and mutant forms of the Aquifex aeolicus GyrB (aqGyrB) N-terminal domain (NTD).
Representative 2Fo–Fc electron density maps (blue mesh, contoured at 3.0σ) around the catalytic acidic residues in the AMPPNP-bound structures of the aqGyrB NTD. (A) Wild-type, (B) E48A, (C) E48Q, (D) D51A, and (E) D51N. The electron density clearly supports the modeled conformations of Glu48, Asp51, and their substituted residues.
Figure 2—figure supplement 2
Conserved positioning of the catalytic Mg²+ ion in the Aquifex aeolicus MutL (aqMutL) N-terminal domain (NTD) and Aquifex aeolicus GyrB (aqGyrB) NTD.
(A) Close-up view of the ATPase active site of the aqMutL NTD. (B) Close-up view of the ATPase active site of the aqGyrB NTD. (C) Superposition of the active sites of the aqMutL NTD (light pink) and aqGyrB NTD (light blue). Mg²+ ions are shown as magenta and purple spheres and catalytic water molecules as small spheres. AMPPNP molecules and side chains of Asn33 of the aqMutL NTD and Asn52 of the aqGyrB NTD are shown as stick models. These Mg²+-coordinating asparagine residues are adjacent to, but distinct from, the second acidic residues Glu32 in aqMutL and Asp51 in aqGyrB examined in this study. The superposition demonstrates that the catalytic Mg²+ ion occupies essentially the same position in the aqMutL and aqGyrB active sites.
Figure 3 with 1 supplement
Structural basis for the loss and preservation of ATP binding in Aquifex aeolicus GyrB (aqGyrB) N-terminal domain (NTD) mutant forms.
The wild-type and all mutant forms were crystallized in the presence of AMPPNP; however, the E48A mutant form did not bind AMPPNP, whereas the E48Q, D51A, and D51N mutant forms were observed in complex with AMPPNP. (A) Overall structures of the wild-type and E48A mutant aqGyrB NTD monomers. The ATPase active site is indicated by a boxed region. The ATP-lid (residues 105–124) in the wild-type form and the corresponding region in the E48A mutant form are shown in magenta and olive, respectively. (B–F) Enlarged views of the ATPase active site in the wild-type (B), E48A (C), E48Q (D), D51A (E), and D51N (F) forms. Side chains of the residues implicated in the ATP-induced conformational changes (e.g. His122, Gln340, and Lys342), as well as residues 48 and 51, are shown as stick models. AMPPNP is depicted as sticks, and the Mg²+ ion is shown as a green sphere. Hydrogen bonds and ionic interactions are indicated by dashed lines.
Figure 3—figure supplement 1
Structure of the ATPase active site of the E. coli GyrB N-terminal domain (NTD).
Residues corresponding to Glu48, Asp51, His122, Gln340, and Lys342 of aqGyrB are Glu42, Asp45, His116, Gln335, and Lys337, respectively, in E. coli GyrB. Side chains of these residues are shown as stick models together with the bound AMPPNP. Hydrogen bonds and ionic interactions are depicted as dashed lines.
Figure 4
Structures surrounding the conserved acidic residues: (A) E48A, (B) E48Q, (C) D51A, and (D) D51N mutant forms of the Aquifex aeolicus GyrB (aqGyrB) N-terminal domain (NTD).
AMPPNP and the side chains at the positions 48 and 51 are shown as stick models. Oxygen atoms are shown in red, nitrogen atoms in blue, and phosphorus atoms in orange. The magnesium ion is shown as a green sphere, and the water molecule proposed to act as the nucleophilic water is shown as a red sphere. Blue mesh represents Fo–Fc omit electron density maps contoured at 3σ, calculated after omission of AMPPNP, the magnesium ion, and the nucleophilic water molecule. Hydrogen bonds are indicated by dashed lines, with the corresponding distances shown.
Figure 5
ATPase activities of human MutL homolog N-terminal domains (NTDs).
(A) ATPase activity of mutant forms of the human Postmeiotic Segregation Increased 2 (PMS2) NTD. (B) ATPase activity of a mutant form of the human MLH1 NTD. ATPase activities were measured using the same procedures as described for Figures 1 and 2. Apparent rate constants (kapp) were plotted as a function of substrate concentration. Data points represent the mean values from three independent measurements, with error bars indicating standard deviations. Solid lines represent the theoretical Michaelis-Menten curves.
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Figure 5—source data 1
This source data file contains the ATPase assay data showing apparent turnover rates (kapp) as a function of ATP concentration for wild-type and mutant human PMS2 N-terminal domains shown in Figure 5A.
- https://cdn.elifesciences.org/articles/111443/elife-111443-fig5-data1-v1.xlsx
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Figure 5—source data 2
This source data file contains the ATPase assay data showing kapp as a function of ATP concentration for wild-type and mutant human MLH1 NTDs shown in Figure 5B.
- https://cdn.elifesciences.org/articles/111443/elife-111443-fig5-data2-v1.xlsx
Figure 6 with 1 supplement
Maximum-likelihood phylogeny of the ATPase domain of representative GHKL family members.
The state of the amino acid residue at the position corresponding to Aquifex aeolicus MutL (aqMutL) Glu32 or aqGyrB Asp51 is indicated by colored bars (red, acidic residue; blue, non-acidic residue). The phylogenetic tree was visualized and annotated using the Interactive Tree of Life (iTOL) web server (Letunic and Bork, 2024). The tree is displayed in a rectangular layout for clarity and should be interpreted as unrooted. Ancestral state reconstruction was performed on the fixed topology, and posterior probabilities are shown for selected internal nodes. The internal node uniting the MutL/GyrB/MORC/Hsp90 clades strongly supports an acidic residue with the posterior probability (PP) of 0.983, whereas the Hsp90 stem ancestor is strongly supported to encode serine at this position with the posterior probability of 1.000. Species abbreviations used in the phylogenetic tree are as follows: Ce, Caenorhabditis elegans; Sc, Saccharomyces cerevisiae; Dr, Danio rerio; Hs, Homo sapiens; Aa, Aquifex aeolicus; Hp, Helicobacter pylori; Tt, Thermus thermophilus; Ec, Escherichia coli; Mt, Mycobacterium tuberculosis; Bs, Bacillus subtilis; Sm, Streptococcus mutans; Dm, Drosophila melanogaster; Clf, Canis lupus familiaris; Mm, Mus musculus; Hc, Hemicordylus capensis; Pv, Patella vulgata; Tf, Tachysurus fulvidraco; Lk, Lepidochelys kempii; Cc, Castor canadensis; Dn, Dasypus novemcinctus.
Figure 6—figure supplement 1
Maximum-likelihood phylogeny corresponding to Figure 6 with branch lengths proportional to the inferred evolutionary distances.
The topology is identical to that shown in Figure 6, whereas branch lengths are proportional to the evolutionary distances inferred by IQ-TREE. The tree was visualized using the Interactive Tree of Life (iTOL) web server. Branch lengths are proportional to the evolutionary distances inferred by IQ-TREE. The scale bar represents an evolutionary distance of one amino acid substitution per site. Species abbreviations are the same as those in Figure 6.
Tables
Table 1
Kinetic parameters for the ATPase activity of the Aquifex aeolicus MutL (aqMutL) N-terminal domains (NTDs), Aquifex aeolicus GyrB (aqGyrB) NTDs, ProS2-tagged human Postmeiotic Segregation Increased 2 (PMS2) NTDs, and histidine-tagged human MLH1 NTDs.
| Proteins | kcat (min–1)* | Km (mM)* | kcat/Km (M–1 s–1) |
|---|---|---|---|
| aqMutL WT | 0.65±0.10 | 0.14±0.026 | 77 |
| E29A | N.D.† | N.D.† | N.D.† |
| E29V | N.D.† | N.D.† | N.D.† |
| E29K | N.D.† | N.D.† | N.D.† |
| E29Q | 0.069±0.010 | 1.5±0.20 | 0.77 |
| E29G | N.D.† | N.D.† | N.D.† |
| E32A | 0.10±0.022 | 2.1±0.39 | 0.79 |
| E32V | 0.15±0.057 | 5.5±1.3 | 0.45 |
| E32K | N.D.† | N.D.† | N.D.† |
| E32Q | 0.10±0.018 | 3.5±0.46 | 0.48 |
| E32G | N.D.† | N.D.† | N.D.† |
| E29Q/E32Q | N.D.† | N.D.† | N.D.† |
| aqGyrB NTD WT | 2.4±0.19 | 0.35±0.066 | 110 |
| E48A | N.D.† | N.D.† | N.D.† |
| E48Q | 0.78±0.16 | 0.75±0.34 | 13 |
| D51A | 0.39±0.024 | 0.21±0.038 | 31 |
| D51N | 1.2±0.18 | 0.28±0.059 | 137 |
| E48Q/D51N | N.D.† | N.D.† | N.D.† |
| human PMS2 NTD WT | 0.62±0.049 | 0.51±0.14 | 20 |
| E44Q | 0.13±0.011 | 0.22±0.079 | 9.8 |
| E44V | 0.085±0.069 | 0.67±0.14 | 2.1 |
| human MLH1 NTD WT | 0.12±0.046 | 0.66±0.064 | 3.0 |
| E37K | N.D.† | N.D.† | N.D.† |
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*
The standard Michaelis-Menten equation was fitted to the data to determine the kcat and Km values.
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†
N.D. means that the activity was not detected.
Table 2
Data collection and refinement statistics for the Aquifex aeolicus GyrB (aqGyrB) N-terminal domains (NTDs).
| Parameters | Wild-type | E48A | E48Q | D51A | D51N |
|---|---|---|---|---|---|
| Data collection | |||||
| Beamline | SPring-8 BL45XU | SPring-8 BL45XU | SPring-8 BL45XU | SPring-8 BL45XU | SPring-8 BL45XU |
| Detector | Dectris PILATUS 6 M | Dectris PILATUS 6 M | Dectris PILATUS 6 M | Dectris PILATUS 6 M | Dectris PILATUS 6 M |
| Wavelength (Å) | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 |
| Exposure time (s) | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 |
| Camera distance (mm) | 140 | 140 | 140 | 140 | 140 |
| Oscillation angle (∘) | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 |
| Oscillation range (∘) | 360 | 360 | 360 | 360 | 360 |
| Space group | P22121 | P6422 | P22121 | P22121 | P22121 |
| Cell dimensions | |||||
| a, b, c (Å) | 78.9, 89.3, 126.3 | 173.1, 173.1, 80.1 | 74.5, 89.0, 131.3 | 75.2, 90.0, 131.7 | 70.0, 91.2, 131.1 |
| α, β, γ (∘) | 90.0, 90.0, 90.0 | 90.0, 90.0, 120.0 | 90.0, 90.0, 90.0 | 90.0, 90.0, 90.0 | 90.0, 90.0, 90.0 |
| Resolution (Å) | 43.16–1.65 (1.71–1.65) * | 46.54–2.66 (2.76–2.66) | 44.50–1.70 (1.76–1.70) | 45.03–1.63 (1.70–1.63) | 39.41–2.05 (2.12–2.05) |
| I/σI | 4.8 (1.3) | 53.4 (15.8) | 9.4 (1.3) | 4.8 (1.4) | 9.4 (1.4) |
| CC1/2 (%) | 99.0 (78.4) | 99.2 (96.4) | 99.7 (50.1) | 96.4 (79.5) | 99.5 (52.9) |
| Rmeas (%) | 2.3 (57.4) | 20.9 (209.6) | 18.4 (174.0) | 22.8 (45.2) | 20.7 (152.7) |
| Completeness (%) | 99.3 (98.4) | 100.0 (100.0) | 99.9 (99.8) | 99.6 (98.4) | 99.9 (99.9) |
| Redundancy | 8.1 (8.0) | 14.2 (14.6) | 7.1 (7.2) | 4.1 (2.6) | 7.0 (6.9) |
| Mosaicity (∘) | 0.39 | 0.31 | 0.10 | 0.46 | 0.25 |
| Refinement | |||||
| No. of reflections | 107387 | 15381 | 96451 | 111409 | 53318 |
| Rwork/Rfree | 0.199/0.216 | 0.242/0.266 | 0.186/0.215 | 0.199/0.233 | 0.205/0.248 |
| No. of atoms | |||||
| Protein | 5660 | 2391 | 5766 | 5860 | 5758 |
| Ligand/Ion | 88 | 11 | 80 | 98 | 77 |
| Water | 285 | 22 | 588 | 227 | 379 |
| B-factor | |||||
| Protein | 34.1 | 68.6 | 25.0 | 35.5 | 29.5 |
| Ligand/Ion | 24.8 | 114.1 | 25.0 | 28.1 | 22.2 |
| Water | 34.6 | 68.4 | 33.4 | 34.4 | 30.6 |
| r.m.s. deviations † | |||||
| Bond lengths (Å) | 0.007 | 0.008 | 0.025 | 0.007 | 0.006 |
| Bond angles (∘) | 0.89 | 1.04 | 1.72 | 1.02 | 0.91 |
| Ramachandran plot | |||||
| Most favored (%) | 97.5 | 95.4 | 98.2 | 97.7 | 97.5 |
| Additional allowed (%) | 2.2 | 4.3 | 1.8 | 1.9 | 2.1 |
| Generously allowed (%) | 0.3 | 0.2 | 0 | 0.4 | 0.4 |
| Disallowed (%) | 0 | 0.1 | 0 | 0 | 0 |
| Protein Data Bank Code | 23 UL | 23UV | 23 UX | 23UY | 23UZ |
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*
Values of the highest resolution shells.
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†
Root mean square deviations.
Additional files
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Supplementary file 1
Primers used for site-directed mutagenesis in this study.
- https://cdn.elifesciences.org/articles/111443/elife-111443-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/111443/elife-111443-mdarchecklist1-v1.docx
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Two Glu/Asp residues cooperatively mediate an early step of ATP hydrolysis in GHKL ATPases MutL and GyrB
eLife 15:RP111443.
https://doi.org/10.7554/eLife.111443.4
