Two Glu/Asp Residues Cooperatively Mediate an Early Step of ATP Hydrolysis in GHKL ATPases MutL and GyrB

Reviewer #1 (Public review):

Summary:

In this manuscript the applicants 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.

In the manuscript, 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, that 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 catalytic 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:

- 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?

The authors need to discuss this issue explicitly to make it clear that conservation of the mechanism is not complete and that other interpretations are possible.

- The structure figures all have omit maps for just the AMPPnP and the water, whereas the density for the the acidic residues and their mutants are not shown.

This has been addressed.

There are some issues with figure S2B and S5.

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 reduce ATPase activity. Activity was abolished when both residues (E29Q/E32Q) are 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 any effect of disease-linked mutations in GHKL ATPases would have strengthened this study.

(3) The effect of other aqMutL NTD E32 mutants, particularly, the E32K mutant on ATP binding remains unclear, although experimental assessment of nucleotide binding would be challenging due to the high protein concentrations required for the equilibrium dialysis assay.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

(1) A major question that remains is why the mutations have so much more detrimental effect in MutL (100-fold lower k_cat/K_M) than they do in GyrB (3-fold lower). Can the authors explain this? Doesn't this argue against the proposed catalytic conservation?

We agree that the quantitative effects of the mutations differ between MutL and GyrB. However, we do not think that this difference argues against conservation of the catalytic mechanism. The trends of the mutational effects are highly consistent between the two enzymes. In both proteins, replacement of the conserved catalytic glutamate with Ala (E29A in aqMutL and E48A in aqGyrB) abolished ATPase activity and ATP binding, whereas replacement with the isosteric amide residue (E29Q and E48Q), which preserves hydrogen-bonding capability but lacks proton-accepting capacity, retained ATP binding and measurable ATPase activity. Likewise, substitutions of the second acidic residue (E32Q in aqMutL and D51N in aqGyrB) also retained substantial ATPase activity despite the loss of proton-accepting capability. Most importantly, simultaneous substitution of both acidic residues (E29Q/E32Q in aqMutL and E48Q/D51N in aqGyrB) completely abolished ATPase activity in both enzymes. Therefore, although the magnitude of the activity reduction caused by the individual mutations differs between MutL and GyrB, the qualitative pattern is essentially identical. We therefore think that the proposed catalytic mechanism is conserved, while the quantitative differences likely reflect differences in the local catalytic environment rather than differences in the underlying mechanism.

(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.

We have added Supplementary Fig. S2, which shows the 2F_o–F_c electron density maps around residues Glu48/Asp51 (or their substituted residues) in the wildtype and all mutant structures. The following sentences have been added in the revised manuscript:

“To show that the introduced substitutions were unambiguously supported by the crystallographic data, electron density maps around residues 48 and 51 are shown in Supplementary Fig. S2.” (p. 4 line 198-200 in the revised manuscript)

Reviewer #2 (Public review):

(1) 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.

We agree that extending the analysis to additional disease-associated variants in other human GHKL ATPases would further strengthen our understanding of the conserved catalytic mechanism and its clinical relevance. However, we believe that such a comprehensive analysis is beyond the scope of the present study, which focuses on establishing the fundamental catalytic mechanism shared between MutL and GyrB. We consider systematic functional and structural analyses of disease-associated variants across the GHKL ATPase family to be an important direction for future research. We have now added a statement to the Results and Discussion section to acknowledge this limitation and highlight this future perspective:

“Although we focused here on pathogenic variants in the MutL homologs MLH1 and PMS2, extending similar structural and biochemical analyses to disease-associated variants in other human GHKL ATPases will be important for evaluating the generality and clinical relevance of the conserved catalytic mechanism proposed in this study.” (p. 6 line 303-306 in the revised manuscript)

(2) 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.

We agree that the complete loss of ATPase activity caused by the MLH1 E37K variant cannot be explained solely by loss of the catalytic carboxylate. However, we note that the corresponding aqMutL E32K variant analyzed in this study also exhibited essentially no detectable ATPase activity, indicating that this phenotype is not unique to MLH1. It can be thought that the severe defect of the lysine variants arises not merely from loss of the acidic side chain but from charge reversal. We have clarified this point in the Results and Discussion sections:

“In contrast, the E37K mutation in the MLH1 NTD completely abolished the ATPase activity under our assay conditions (Fig. 5B and Table 1) unlike the corresponding glutamine substitutions, which retained substantial residual ATPase activity in aqMutL, aqGyrB, and PMS2 NTDs. A similar complete loss of ATPase activity was also observed for the E32K mutant form of the aqMutL NTD. These observations suggest that the severe defect caused by the lysine substitution cannot be attributed simply to loss of the catalytic carboxylate. Instead, introduction of a positively charged side chain (charge reversal) is likely to perturb the local electrostatic environment. Structural characterization of the MLH1 E37K and aqMutL E32K mutant forms will be required to clarify the molecular basis of this severe functional defect.” (p. 6 line 281-289 in the revised manuscript)

(3) 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.

We performed additional ATP-binding experiments using the aqMutL NTD E32A and aqGyrB NTD D51A mutant forms. Both mutant forms exhibited ATP-binding activities comparable to those of the corresponding wildtype forms. These results support our conclusion that the second acidic residue primarily contributes to ATP hydrolysis rather than ATP binding, whereas the first acidic residue plays dual roles in ATP binding and catalysis.

Although we agree that nucleotide-binding analyses of the MLH1 and PMS2 variants would be informative, these experiments were not feasible because the equilibrium dialysis assay requires high protein concentrations, which we were unable to obtain for the recombinant human MLH1 and PMS2 N-terminal domains.

We have incorporated these new data into the Results and Discussion section:

“In contrast to the E29A mutant form of the aqMutL NTD, the E32A mutant form exhibited ATP binding ability comparable to that of the wildtype form (Supplementary Fig. S1A), indicating that Glu32 does not contribute to ATP binding.” (p. 3 line 143-145 in the revised manuscript)

“The D51A mutant form of the aqGyrB NTD retained ATP binding ability comparable to that of the wildtype form, indicating that Asp51 is not required for nucleotide binding (Supplementary Fig. S1B).” (p. 4 line 183-185 in the revised manuscript)

(4) 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.

We thank the reviewer for raising this important point. We agree that the possible influence of the purification/solubility tags on the absolute ATPase activities of the PMS2 and MLH1 NTDs cannot be completely excluded. However, the wild-type and mutant forms for each homolog were analyzed using identical constructs under the same experimental conditions. Therefore, the affinity/solubility tags are unlikely to affect the relative comparisons of the mutational effects. Furthermore, because the affinity tags are located at the N terminus and are distant from the ATPase active site, they are unlikely to directly perturb the catalytic center.

(5) 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.

We thank the reviewer for this insightful comment. Our proposed mechanism is based on the cooperative catalytic roles of the two conserved acidic residues, namely the involvement of the first acidic residue in ATP binding and nucleophilic water positioning and the role of the second acidic residue in proton abstraction. In contrast, the Glu48–Gln340 hydrogen-bonding interaction described in aqGyrB was proposed only as a structural feature that may modulate the contribution of the first acidic residue to ATP binding. It is not an essential component of the catalytic mechanism proposed in this study. Therefore, the absence of this particular hydrogen-bonding network in the currently available structures of MutL and MORC proteins does not argue against conservation of the catalytic mechanism itself.

Recommendations for the authors:

Reviewing Editor Comments:

One of the structures (Crystal Structure of the E48A variant) has relatively poor statistics in the PDB validation report. Please improve this structure.

We performed additional refinement of the E48A crystal structure. This resulted in a clear improvement in the overall model quality, with the Ramachandran favored residues increasing from 93.4% to 95.4%, the percentage of side-chain outliers decreasing from 6.1% to 1.4%. The refined structural model has been used throughout the revised manuscript, and the updated refinement statistics are provided in Table 2.

Reviewer #1 (Recommendations for the authors):

Please show conventional density maps (e.g., sigmaA weighted 2fo-fc maps).

This comment is closely related to Comment (2) in the Public Review by the Reviewer #1. In response, we have added Supplementary Fig. S2, which presents conventional σA-weighted 2F_o–F_c electron density maps around the catalytic acidic residues in the wild-type and mutant aqGyrB structures.

Reviewer #2 (Recommendations for the authors):

(1) Regarding the analysis of clinical variants, it would be informative to note that the second allele is lost before tumor growth in Lynch syndrome.

“Therefore, these variants might contribute to the development of Lynch syndrome by weakening the ATPase-driven regulatory functions of MutL.” (p. 6 line 280-281 in the original manuscript) has been changed to:

“In individuals carrying these germline variants, subsequent loss or inactivation of the remaining wildtype allele would leave only the ATPase-defective MutL protein, thereby compromising mismatch repair and promoting tumorigenesis.” (p. 6 line 296-298 in the revised manuscript)

(2) P. 4, in the paragraph "Conserved roles of two acidic residues of aqGyrB in ATP hydrolysis", the E48Q mutant retains approximately one third of the WT activity, not one quarter as stated in the text (Table 1). Additionally, later in the article, the D51 mutant is reported to retain approximately one-sixth (~17%) of the WT activity, rather than ~25% as written.

We thank the reviewer for carefully identifying these inconsistencies. The text has been corrected to accurately reflect the data presented in Table 1: “…one third of the wildtype activity” (p. 4 line 180) and “…retaining ~16%...” (p. 4 line 186 in the revised manuscript)

(3) P. 6, lines 275-276, this sentence should be rephrased for clarity, as the authors note at the end of page 5 that not all members of the GHKL ATPase family possess this second acidic residue.

“…this second acidic residue plays a conserved and functionally significant role in ATP hydrolysis across the GHKL ATPase family.” in the original manuscript has been changed to:

“…this second acidic residue plays a conserved and functionally significant role in ATP hydrolysis among some members of the GHKL ATPase family.” (p. 6 line 292 in the revised manuscript)

(4) P. 9, in the "Data Accessibility Statement", the PDB code 23UY is missing. This entry corresponds to the crystal structure of the D51A mutant of aqGyrB NTD and should be included.

The Data Accessibility Statement has been revised to include the code 23UY. (p. 9 line 454 in the revised manuscript)

(5) P. 14, the table should be labelled "Table 2. Data collection and refinement statistics for the aqGyrB NTDs", rather than "Supplementary Table 2", to ensure consistency with how it is cited in the main text.

The table title has been corrected from "Supplementary Table 2" to "Table 2”. (p. 4 line 198 in the revised manuscript)

(6) It is difficult to determine from the figures whether the magnesium ion is positioned equivalently in aqMutL and aqGyrB. Did the authors observe any differences in ion positioning?

To facilitate direct comparison of the catalytic Mg²⁺ ion between the aqMutL and aqGyrB NTDs, we have added Supplementary Fig. S3, which shows a structural superimposition of the ATPase active sites of the two proteins:

“Structural superposition of the aqGyrB NTD and aqMutL NTD revealed that the catalytic Mg²⁺ ion occupies essentially the same position in the two ATPase active sites (Supplementary Fig. S3), indicating that the metal-binding geometry is highly conserved, where the Mg²⁺ ion is coordinated by the side chain of the conserved Asn, AMPPNP, and surrounding water molecules. Neither Glu48 of aqMutL nor Asp51 of aqGyrB directly coordinated the Mg²⁺ ion.” (p. 5 line 201-205 in the revised manuscript)

(7) The authors should discuss the interaction between the aqGyrB NTD, Mg²⁺, and ATP during the binding step. In the case of the E48A mutant, where ATP binding is lost, does E48 directly establish contacts with Mg²⁺, or is another residue involved (with conformational changes preventing this interaction)?

Our structural analyses indicate that Glu48 does not directly coordinate the catalytic Mg²⁺ ion. Instead, as shown in Supplementary Fig. S3, the Mg²⁺ ion is coordinated by the side chain of Asn52, AMPPNP, and surrounding water molecules. We have clarified this point in the Results and Discussion sections:

“Structural superposition of the aqGyrB NTD and aqMutL NTD revealed that the catalytic Mg²⁺ ion occupies essentially the same position in the two ATPase active sites (Supplementary Fig. S3), indicating that the metal-binding geometry is highly conserved, where the Mg²⁺ ion is coordinated by the side chain of Asn52, AMPPNP, and surrounding water molecules. Neither Glu48 nor Asp51 directly coordinated the Mg²⁺ ion.” (p. 5 line 201-205 in the revised manuscript)

(8) Figures 1 and 3: Use ribbon representation and no shadows, at least for the inset panels, to enhance clarity and interpretability.

We have revised Figures 1 and 3 by displaying the protein structures in ribbon representation and removing shadows from the inset panels.

(9) Combine Figures 1 and 2, and combine Figures 3 and 4.

Following the reviewer's recommendation, we have combined the original Figures 1 and 2 into a single figure and the original Figures 3 and 4 into another single figure.

(10) Figure 5: Zoom in further and remove shadows. The current panels are not very effective in highlighting how ATP is bound by the different protein variants.

Figure 5 has been revised by increasing the magnification of the ATP-binding sites and removing shadows from the structural renderings.

(11) Figure 8. Add a scale bar to show evolutionary distance.

We thank the reviewer for this helpful suggestion. To provide information on evolutionary distances while preserving the clarity of the main figure, we have added a new Supplementary Fig. S5 showing the same phylogenetic tree with branch lengths proportional to the inferred evolutionary distances and an evolutionary distance scale bar. Figure 6 has been retained in its simplified form with equal branch lengths to facilitate visualization of the ancestral-state reconstruction, and we have clarified this distinction in the Materials and Methods section:

“For visualization purposes, branch lengths were not scaled and were displayed with equal lengths in Fig. 6. The corresponding phylogeny with branch lengths proportional to the inferred evolutionary distances is provided in Supplementary Fig. S5.” (p. 9 line 429-432 in the revised manuscript)