Allosteric cooperation in β-lactam binding to a non-classical transpeptidase
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Curated by eLife
Evaluation Summary:
This manuscript reports high-resolution crystallographic structures of the L,D, transpeptidase from Mycobacterium tuberculosis. These structures were obtained with ligands (a sugar molecule and a beta-lactam). A surprising finding is that the enzyme contains a ligand-binding site located greater than 20 Å away from the catalytic site. The authors propose and provide some evidence for an allosteric role of the new ligand site (S-pocket), which would be significant because it could allow new ways of targeting the protein for inhibition. While enthusiasm is high for the discovery of a putative allosteric site, more rigorous computation is necessary, along with some biochemical investigations and mutagenesis studies to rule out the possibility of a different role for the S-site. Moreover, a better articulation of the connection/crosstalk between the two sites in the form of a mechanistic hypothesis would strengthen the paper.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
L,D -transpeptidase function predominates in atypical 3 → 3 transpeptide networking of peptidoglycan (PG) layer in Mycobacterium tuberculosis . Prior studies of L,D -transpeptidases have identified only the catalytic site that binds to peptide moiety of the PG substrate or β-lactam antibiotics. This insight was leveraged to develop mechanism of its activity and inhibition by β-lactams. Here, we report identification of an allosteric site at a distance of 21 Å from the catalytic site that binds the sugar moiety of PG substrates (hereafter referred to as the S-pocket). This site also binds a second β-lactam molecule and influences binding at the catalytic site. We provide evidence that two β-lactam molecules bind co-operatively to this enzyme, one non-covalently at the S-pocket and one covalently at the catalytic site. This dual β-lactam-binding phenomenon is previously unknown and is an observation that may offer novel approaches for the structure-based design of new drugs against M. tuberculosis .
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Evaluation Summary:
This manuscript reports high-resolution crystallographic structures of the L,D, transpeptidase from Mycobacterium tuberculosis. These structures were obtained with ligands (a sugar molecule and a beta-lactam). A surprising finding is that the enzyme contains a ligand-binding site located greater than 20 Å away from the catalytic site. The authors propose and provide some evidence for an allosteric role of the new ligand site (S-pocket), which would be significant because it could allow new ways of targeting the protein for inhibition. While enthusiasm is high for the discovery of a putative allosteric site, more rigorous computation is necessary, along with some biochemical investigations and mutagenesis studies to rule out the possibility of a different role for the S-site. Moreover, a better articulation of the …
Evaluation Summary:
This manuscript reports high-resolution crystallographic structures of the L,D, transpeptidase from Mycobacterium tuberculosis. These structures were obtained with ligands (a sugar molecule and a beta-lactam). A surprising finding is that the enzyme contains a ligand-binding site located greater than 20 Å away from the catalytic site. The authors propose and provide some evidence for an allosteric role of the new ligand site (S-pocket), which would be significant because it could allow new ways of targeting the protein for inhibition. While enthusiasm is high for the discovery of a putative allosteric site, more rigorous computation is necessary, along with some biochemical investigations and mutagenesis studies to rule out the possibility of a different role for the S-site. Moreover, a better articulation of the connection/crosstalk between the two sites in the form of a mechanistic hypothesis would strengthen the paper.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript by Ahmad et al. reports on the high-resolution crystal structures of sugar and a β-lactam molecule binding to an allosteric site of Mycobacterium tuberculosis L,D-transpeptidase (LdtMt2). The structural changes upon dual ligand binding highlight the structural alterations that span across the allosteric pocket and the catalytic site. The authors systematically mutate residues in both, the allosteric site and the catalytic site and use biochemical assays to show how they are linked.
Strengths:
Structural, biochemical and Mutagenesis experiments
(a) High-resolution crystal structures of LdtMt2 with ligands in both allosteric site and orthosteric site.
(b) The study unequivocally demonstrates that residue S351 forms a part of the catalytic triad and not S337, as previously reported in the …Reviewer #1 (Public Review):
The manuscript by Ahmad et al. reports on the high-resolution crystal structures of sugar and a β-lactam molecule binding to an allosteric site of Mycobacterium tuberculosis L,D-transpeptidase (LdtMt2). The structural changes upon dual ligand binding highlight the structural alterations that span across the allosteric pocket and the catalytic site. The authors systematically mutate residues in both, the allosteric site and the catalytic site and use biochemical assays to show how they are linked.
Strengths:
Structural, biochemical and Mutagenesis experiments
(a) High-resolution crystal structures of LdtMt2 with ligands in both allosteric site and orthosteric site.
(b) The study unequivocally demonstrates that residue S351 forms a part of the catalytic triad and not S337, as previously reported in the literature.
(c) Residue R209 in the allosteric pocket is directly linked to the catalytic activity.Weakness:
Computational studies
(a) How exactly are the two sites structurally linked across a distance of >20Å is not explained. -
Reviewer #2 (Public Review):
In this work, authors seek a better understanding of the L,D-transpeptidase class of enzymes. They investigate Ldt-Mt2, 1 of five L,D-transpeptidase paralogs rom Mycobacterium tuberculosis. They determine a crystal structure of LdtMt2 and identify a pocket between two domains YkuD and IgD2. This pocket is hereafter referred to as the S-pocket. In summary, authors conclude that the S-pocket is an allosteric site that controls catalytic activity of Ldt-Mt2.
This study is potentially very exciting and very important, as the report of this allosteric site is novel and might be a prevailing mechanism across other L,D-transpeptidases.
There are two major issues that should be addressed. 1) Because of the nature of the evidence presented to support the existence/role of the S-pocket (i.e. computational modeling, …
Reviewer #2 (Public Review):
In this work, authors seek a better understanding of the L,D-transpeptidase class of enzymes. They investigate Ldt-Mt2, 1 of five L,D-transpeptidase paralogs rom Mycobacterium tuberculosis. They determine a crystal structure of LdtMt2 and identify a pocket between two domains YkuD and IgD2. This pocket is hereafter referred to as the S-pocket. In summary, authors conclude that the S-pocket is an allosteric site that controls catalytic activity of Ldt-Mt2.
This study is potentially very exciting and very important, as the report of this allosteric site is novel and might be a prevailing mechanism across other L,D-transpeptidases.
There are two major issues that should be addressed. 1) Because of the nature of the evidence presented to support the existence/role of the S-pocket (i.e. computational modeling, ligands modeled into electron density, a single disruptive mutation), it would be helpful for the authors to provide an additional piece of data to support the existence of the S-pocket. 2) The computational studies as currently presented, are not rigorous enough to support the claims that are made.
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Reviewer #3 (Public Review):
This study reports on a novel site termed as the 'S-pocket' that is located at a hinge region of the LdtM2 transpeptidase of M. tuberculosis. Building on observations of the presence of a glucose molecule within the crystal structure at this site, the authors hypothesise that this may also serve as a binding site for peptidoglycan precursors such as NAG and model a pentameric peptidoglycan molecule across the S-pocket and the inner cavity of the catalytic site. However, Thermofluor data of the effect of NAG binding is weak. It would be helpful for the readers to compare this data with similar data for glucose. Additional data for other point mutations within the S-pocket as well as a point mutation in the active site (e.g. at the inner cavity) will be needed to fully justify these conclusions.
The remaining …
Reviewer #3 (Public Review):
This study reports on a novel site termed as the 'S-pocket' that is located at a hinge region of the LdtM2 transpeptidase of M. tuberculosis. Building on observations of the presence of a glucose molecule within the crystal structure at this site, the authors hypothesise that this may also serve as a binding site for peptidoglycan precursors such as NAG and model a pentameric peptidoglycan molecule across the S-pocket and the inner cavity of the catalytic site. However, Thermofluor data of the effect of NAG binding is weak. It would be helpful for the readers to compare this data with similar data for glucose. Additional data for other point mutations within the S-pocket as well as a point mutation in the active site (e.g. at the inner cavity) will be needed to fully justify these conclusions.
The remaining bulk of the manuscript is devoted to understanding the interaction of beta-lactams with the S-pocket. The authors present convincing Thermofluor data to demonstrate a clear synergy between non-covalent interactions at the S-pocket and covalent binding at the catalytic site. However, the MD simulations have not been performed with the same rigour and at this stage, do not add to the strength of the evidence presented. Furthermore, inclusion of animations or videos from the MD simulations performed for this study would significantly help the readers grasp these points in an intuitive fashion versus the snapshots presented here. The authors directly apply this knowledge to understanding the interaction of an experimental carbapenem T203 with the M. tuberculosis Ld transpeptidase, showcasing how this improved understanding of the functioning of LdT transpeptidases in this pathogen can translate to developing new tools to combat this globally relevant pathogen.
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