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

Grabowska M, Jagielska E, Czapinska H, Bochtler M, Sabala I - Sci Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology, Ks. Trojdena 4, 02-109 Warsaw, Poland.

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.

No MeSH data available.


Related in: MedlinePlus

Peptides and analogues in the substrate binding groove of LytM.(A) LytM catalytic domain with the phosphinate transition state analogue; (B) full length LytM with active site blocked by peptide from occluding region (PDB 1QWY); (C,D) catalytic domain of LytM with glycine rich loop from neighboring molecule wedged into the active site cleft (P41 (2B13) and P3221 (2B44) crystal forms). Arrows mark the direction of the main chain in the transition state analogue and protein fragments occupying in the cleft.
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f5: Peptides and analogues in the substrate binding groove of LytM.(A) LytM catalytic domain with the phosphinate transition state analogue; (B) full length LytM with active site blocked by peptide from occluding region (PDB 1QWY); (C,D) catalytic domain of LytM with glycine rich loop from neighboring molecule wedged into the active site cleft (P41 (2B13) and P3221 (2B44) crystal forms). Arrows mark the direction of the main chain in the transition state analogue and protein fragments occupying in the cleft.

Mentions: LytM happens to contain a surface exposed loop distant from the active site with 3 consecutive glycine residues (Gly206-Gly208). In two previous crystal forms, this loop comes close to the active site of another LytM molecule, so that it might mimic the binding of a substrate6. However, it was already noticed in the previous work that the glycine-rich loop was running through the active site region in opposite directions in different crystal forms (Fig. 5). Moreover, the resolution was sufficient to conclude that the loop was not cleaved in the “incidental” crystallographic contacts, and therefore could not mimic the binding mode of a substrate accurately6. We have now revisited the previously observed binding modes in the light of the experimental density for the phosphinate. The glycine rich loop of a neighboring molecule in the P41 crystal form (PDB: 2B13) runs indeed through the region of the active site cleft also occupied by the well-defined part of the phosphinate, but the direction of the polypeptide chain is reversed (Fig. 5C). The loop from the neighboring molecule in the P3221 crystal form (PDB: 2B44) runs in the correct direction, but is very significantly displaced relative to the phosphinate (Fig. 5D). We conclude from this analysis that the crystallographic packing effects that were noticed earlier did correctly identify the active site cleft, but did not accurately mimic the substrate binding mode.


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

Grabowska M, Jagielska E, Czapinska H, Bochtler M, Sabala I - Sci Rep (2015)

Peptides and analogues in the substrate binding groove of LytM.(A) LytM catalytic domain with the phosphinate transition state analogue; (B) full length LytM with active site blocked by peptide from occluding region (PDB 1QWY); (C,D) catalytic domain of LytM with glycine rich loop from neighboring molecule wedged into the active site cleft (P41 (2B13) and P3221 (2B44) crystal forms). Arrows mark the direction of the main chain in the transition state analogue and protein fragments occupying in the cleft.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4594094&req=5

f5: Peptides and analogues in the substrate binding groove of LytM.(A) LytM catalytic domain with the phosphinate transition state analogue; (B) full length LytM with active site blocked by peptide from occluding region (PDB 1QWY); (C,D) catalytic domain of LytM with glycine rich loop from neighboring molecule wedged into the active site cleft (P41 (2B13) and P3221 (2B44) crystal forms). Arrows mark the direction of the main chain in the transition state analogue and protein fragments occupying in the cleft.
Mentions: LytM happens to contain a surface exposed loop distant from the active site with 3 consecutive glycine residues (Gly206-Gly208). In two previous crystal forms, this loop comes close to the active site of another LytM molecule, so that it might mimic the binding of a substrate6. However, it was already noticed in the previous work that the glycine-rich loop was running through the active site region in opposite directions in different crystal forms (Fig. 5). Moreover, the resolution was sufficient to conclude that the loop was not cleaved in the “incidental” crystallographic contacts, and therefore could not mimic the binding mode of a substrate accurately6. We have now revisited the previously observed binding modes in the light of the experimental density for the phosphinate. The glycine rich loop of a neighboring molecule in the P41 crystal form (PDB: 2B13) runs indeed through the region of the active site cleft also occupied by the well-defined part of the phosphinate, but the direction of the polypeptide chain is reversed (Fig. 5C). The loop from the neighboring molecule in the P3221 crystal form (PDB: 2B44) runs in the correct direction, but is very significantly displaced relative to the phosphinate (Fig. 5D). We conclude from this analysis that the crystallographic packing effects that were noticed earlier did correctly identify the active site cleft, but did not accurately mimic the substrate binding mode.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology, Ks. Trojdena 4, 02-109 Warsaw, Poland.

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.

No MeSH data available.


Related in: MedlinePlus