Two Glu/Asp Residues Cooperatively Mediate an Early Step of ATP Hydrolysis in GHKL ATPases MutL and GyrB
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eLife Assessment
In this solid work, Fukui et al. re-examined the ATP hydrolysis mechanism in GHKL ATPases, revealing a cooperative role for two conserved acidic residues rather than a single one. This useful study used a range of biochemical and structural techniques on various mutants from different members of the GHKL ATPase family to test and validate their proposed mechanism. An updated and extended mechanistic model of ATP hydrolysis by this class of enzymes is proposed.
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Abstract
GHKL ATPases share a unique Bergerat ATP-binding fold and regulate diverse biological processes through ATP-dependent conformational changes. An early step of ATP hydrolysis in this family has been attributed to a single highly conserved glutamate residue proposed to function as the general base. However, mutations of this residue impair both the ATPase activity and ATP binding, complicating interpretation of its catalytic role. Re-examination of the high-resolution crystal structures revealed a second conserved acidic residue positioned within a hydrogen-bonding distance from the nucleophilic water molecule. Using Aquifex aeolicus MutL and GyrB as model enzymes, we combined systematic mutagenesis, ATPase and ATP-binding assays, and X-ray crystallography to dissect the roles of these residues. We show that alignment of the nucleophilic water can be maintained as long as the conserved glutamate retains hydrogen-bonding capability, whereas efficient ATP hydrolysis requires proton-accepting capacity at least one of the two acidic residues. These results indicate that the conserved glutamate primarily governs positioning of the nucleophilic water, while activation of this water for catalysis is achieved through cooperative general base function of the two acidic residues. Extending this framework to human MutL homologs, PMS2 and MLH1, we showed that clinically reported variants of uncertain significance in these DNA mismatch repair proteins substantially reduced the ATPase activity, indicating functional impairment. Together, our findings refine the catalytic mechanism of GHKL ATPases and provide a structural and functional framework for interpreting disease-associated variants in GHKL ATPases. Phylogenetic and ancestral state analysis further indicated that the second acidic residue was likely to be present in the common ancestor of major GHKL ATPase lineages but was later modified in a branch including Hsp90, suggesting evolutionary remodeling of the catalytic mechanism in the branch.
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eLife Assessment
In this solid work, Fukui et al. re-examined the ATP hydrolysis mechanism in GHKL ATPases, revealing a cooperative role for two conserved acidic residues rather than a single one. This useful study used a range of biochemical and structural techniques on various mutants from different members of the GHKL ATPase family to test and validate their proposed mechanism. An updated and extended mechanistic model of ATP hydrolysis by this class of enzymes is proposed.
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Reviewer #1 (Public review):
Summary:
In this manuscript, the authors study two residues in the GHKL ATPase active site of Aq MutL and GyrB, and argue that the catalytic base function is shared between two conserved acidic residues that are 3 residues apart.
They generated mutant versions in MutL and GyrB (both ala and the appropriate Asn/Gln version) and performed ATPase analysis. They also generated high-resolution crystal structures of the GyrB NTD with AMPPnP for WT and mutants of the two acidic residues. The data show that mutation in either of these residues does not fully kill activity (with the exception of the Alanine mutation of the first of the two, which interferes with ATP (or AMPPnP) binding). When the acidic residues are mutated to Asn/Gln, the catalytic water can still be positioned, and hence these mutants are more …
Reviewer #1 (Public review):
Summary:
In this manuscript, the authors study two residues in the GHKL ATPase active site of Aq MutL and GyrB, and argue that the catalytic base function is shared between two conserved acidic residues that are 3 residues apart.
They generated mutant versions in MutL and GyrB (both ala and the appropriate Asn/Gln version) and performed ATPase analysis. They also generated high-resolution crystal structures of the GyrB NTD with AMPPnP for WT and mutants of the two acidic residues. The data show that mutation in either of these residues does not fully kill activity (with the exception of the Alanine mutation of the first of the two, which interferes with ATP (or AMPPnP) binding). When the acidic residues are mutated to Asn/Gln, the catalytic water can still be positioned, and hence these mutants are more active than the Ala mutants. In both cases, the double mutation is catalytically dead.
The authors then perform phylogenetic analysis and ancestral gene reconstruction, and based on this, they argue that HSP90 forms a different class of GHKL ATPases, and lost rather than gained this separate status.
Strengths:
The biochemical analysis seems solid.
Weaknesses:
(1) A major question that remains is why the mutations have so much more detrimental effect in MutL (100-fold lower kcat/KM) than they do in GyrB (3-fold lower). Can the authors explain this? Doesn't this argue against the proposed catalytic conservation?
(2) The structure figures all have omit maps for just the AMPPnP and the water, whereas the density for the acidic residues and their mutants is not shown.
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Reviewer #2 (Public review):
Summary:
In this manuscript, Fukui et al. re-examined the ATP hydrolysis mechanism in GHKL ATPases, revealing a cooperative role of two conserved acidic residues rather than one. The authors have used a range of biochemical and structural techniques on various mutants from different members of the GHKL ATPase family to test and validate their proposed mechanism.
Through a detailed re-analysis of their previously published structure of the aqMutL NTD (ATPase domain) in complex with AMPPCP, they identified Glu29 and Glu32 as interacting with nucleophilic water for the catalysis. The authors carefully dissected the respective roles of these two acidic residues with a series of site-directed mutations. Mutations at Glu29 impaired ATPase activity without affecting protein secondary structure or ATP binding in the …
Reviewer #2 (Public review):
Summary:
In this manuscript, Fukui et al. re-examined the ATP hydrolysis mechanism in GHKL ATPases, revealing a cooperative role of two conserved acidic residues rather than one. The authors have used a range of biochemical and structural techniques on various mutants from different members of the GHKL ATPase family to test and validate their proposed mechanism.
Through a detailed re-analysis of their previously published structure of the aqMutL NTD (ATPase domain) in complex with AMPPCP, they identified Glu29 and Glu32 as interacting with nucleophilic water for the catalysis. The authors carefully dissected the respective roles of these two acidic residues with a series of site-directed mutations. Mutations at Glu29 impaired ATPase activity without affecting protein secondary structure or ATP binding in the case of the E29Q mutant. Moreover, mutations at Glu32 did not affect secondary structure (except for E32G) but reduced ATPase activity. Activity was abolished when both residues (E29Q/E32Q) were mutated.
The authors extended their study to another GHKL ATPase, aqGyrB. Their findings further supported the cooperative function of the corresponding acidic residues in aqGyrB (Glu48 and Asp51) during ATP hydrolysis. Mutation of these residues partially impaired ATP hydrolysis without affecting protein secondary structure. ATPase activity was completely lost in the double mutant E48Q/D51M. While the E48Q mutant retained the ability to bind ATP, the E48A mutant did not. High-resolution structures of the WT and E48A, E48Q, D51A, and D51N mutants of the aqGyrB NTD demonstrated that nucleophilic water positioning depended on these residues. E48 played a dominant role in water positioning and is critical for stabilising ATP lid formation and associated conformational changes, whereas D51 contributed cooperatively to catalysis.
The authors investigated the functional impact of mutating the corresponding residues in the human MutL homologs PMS2 and MLH1. Clinical variants consistently exhibited reduced or abolished ATPase activity, providing a potential molecular basis for Lynch syndrome through impaired DNA mismatch repair.
Lastly, through evolutionary analysis, the authors inferred that the second acidic residue was likely present in the common ancestor of MutL, GyrB, and MORC proteins, but was lost in the case of Hsp90.
Strengths:
(1) This study contains a detailed structural and biochemical analysis of a biologically important set of GHKL ATPases. The authors identify a second acidic residue that is conserved and contributes to catalysis in a large subset of GHKL ATPases. An updated and extended mechanistic model of ATP hydrolysis by this class of enzymes is proposed, which involves cooperative and partially overlapping roles for the catalytic residue pair. This revised mechanistic model is invaluable for the interpretation of clinical variants of GHKL ATPases such as PMS2 and MLH1.
(2) The work described was performed to an excellent and rigorous technical standard. The structural and biochemical data are sound. The evidence supporting the claims is compelling.
Weaknesses:
(1) The identification in this study of a second acidic residue contributing to catalysis but not absolutely essential for catalysis is a useful finding. However, given that many structures of GHLK ATPases have been determined with different nucleotide analogs bound and that the essential role of the first acidic residue is well established, the importance and scope of the advances described here remain focused within the field of study of GHKL ATPases.
(2) The authors assessed the consequences of variants in the human MutL homologs PMS2 and MLH1, but various other human GHKL ATPases contain clinically relevant variants, some of which have stronger disease associations than the mutations examined in this study. A broader analysis of the effect (or likely effect) of disease-linked mutations in GHKL ATPases would have strengthened this study.
(3) In MLH1, the E37K mutation completely abolishes ATPase activity, but the corresponding mutations in aqMutL, aqGyrB, and PMS2 do not. It remains unclear why E37K in MLH1 leads to complete loss of activity, as the authors propose that water molecule positioning via the first acidic residue, as well as ATP lid stabilisation and associated conformational changes, should still be possible.
(4) The authors do not examine ATP binding in the E32 mutants of aqMutL NTD and the D51 mutants of aqGyrB, or AMPPNP binding of the NLH1 and PMS2 mutants. Hence, the relative contributions of the acidic residues to ATP binding and hydrolysis remain partially unclear.
(5) The ATPase assays for PMS2 and MLH1 (Figure 7 and Table 1) were performed with purification/solubility tags still present. Hence, it cannot be ruled out that these tags influence the measured activities.
(6) The authors suggest that the two-acidic-residue mechanism proposed in this study could be shared among several GHKL ATPase families, yet they also state that the hydrogen-bonding network was not observed in MutL and MORC family proteins. This raises doubt about how conserved the mechanism is, e.g., in MutL and MORC proteins.
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