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High resolution structure of an M23 peptidase with a substrate analogue.

ABSTRACT: LytM is a Staphylococcus aureus autolysin and a homologue of the S. simulans lysostaphin. Both enzymes are members of M23 metallopeptidase family (MEROPS) comprising primarily bacterial peptidoglycan hydrolases. LytM occurs naturally in a latent form, but can be activated by cleavage of an inhibitory N-terminal proregion. Here, we present a 1.45?Å crystal structure of LytM catalytic domain with a transition state analogue, tetraglycine phosphinate, bound in the active site. In the electron density, the active site of the peptidase, the phosphinate and the "diglycine" fragment on the P1' side of the transition state analogue are very well defined. The density is much poorer or even absent for the P1 side of the ligand. The structure is consistent with the involvement of His260 and/or His291 in the activation of the water nucleophile and suggests a possible catalytic role for Tyr204, which we confirmed by mutagenesis. Possible mechanisms of catalysis and the structural basis of substrate specificity are discussed based on the structure analysis.

SUBMITTER: Grabowska M

PROVIDER: S-EPMC4594094 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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High resolution structure of an M23 peptidase with a substrate analogue.

Grabowska Maja M Jagielska Elzbieta E Czapinska Honorata H Bochtler Matthias M Sabala Izabela I

Scientific reports 20151006

LytM is a Staphylococcus aureus autolysin and a homologue of the S. simulans lysostaphin. Both enzymes are members of M23 metallopeptidase family (MEROPS) comprising primarily bacterial peptidoglycan hydrolases. LytM occurs naturally in a latent form, but can be activated by cleavage of an inhibitory N-terminal proregion. Here, we present a 1.45 Å crystal structure of LytM catalytic domain with a transition state analogue, tetraglycine phosphinate, bound in the active site. In the electron densi ...[more]

PMID: 26437833

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Project description:Cysteine proteases are among the most abundant hydrolytic enzymes produced by bacteria, and this diverse family of proteins have significant biological roles in bacterial viability and environmental interactions. Members of the clostripain-like (C11) family of cysteine proteases from commensal gut bacterial strains have recently been shown to mediate immune responses by inducing neutrophil phagocytosis and activating bacterial pathogenic toxins. Development of substrates, inhibitors, and probes that target C11 proteases from enteric bacteria will help to establish the role of these proteins at the interface of the host and microbiome in health and disease. We employed a mass spectrometry-based substrate profiling method to identify an optimal peptide substrate of PmC11, a C11 protease secreted by the commensal bacterium Parabacteroides merdae. Using this substrate sequence information, we synthesized a panel of fluorogenic substrates to calculate kcat and KM and to evaluate the importance of the P2 amino acid for substrate turnover. A potent and irreversible tetrapeptide inhibitor with a C-terminal acyloxymethyl ketone warhead, Ac-VLTK-AOMK, was then synthesized. We determined the crystal structure of PmC11 in complex with this inhibitor and uncovered key active-site interactions that govern PmC11 substrate recognition and specificity. This is the first C11 protease structure in complex with a substrate mimetic and is also the highest resolution crystal structure of a C11 protease to date at 1.12 Å resolution. Importantly, subjecting human epithelial cell lysates to PmC11 hydrolysis in combination with subtiligase-based N-terminal labeling and tandem mass spectrometry proteomics complemented the stringent substrate specificity observed in the in vitro substrate profiling experiment. The combination of chemical biological, biophysical, and biochemical techniques presented here to elucidate and characterize PmC11 substrate selectivity can be expanded to other proteases and the development of chemical tools to study these essential proteins in biologically relevant samples, such as the highly complex distal gut microbiome.

Dataset Information

High resolution structure of an M23 peptidase with a substrate analogue.

Publications

High resolution structure of an M23 peptidase with a substrate analogue.

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