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Crystal structures of Staphylococcus aureus sortase A and its substrate complex.

Yinong Zong1, Todd W Bice, Hung Ton-That

  • 1Center for Biophysical Sciences and Engineering, School of Optometry, University of Alabama, Birmingham, Alabama 35294, USA.

The Journal of Biological Chemistry
|May 1, 2004
PubMed
Summary

This study reveals the crystal structures of Staphylococcus aureus sortase A (SrtA) and its complex with a substrate peptide. SrtA is an enzyme that anchors surface proteins to the bacterial cell wall by cleaving a specific motif in these proteins. The researchers determined the positions of key residues in the active site of SrtA and how they interact with the substrate. They found that conserved residues, including His(120), Cys(184), and Arg(197), are positioned to facilitate the cleavage of the T-G bond in the LPXTG motif. The study also compared the active sites of SrtA and sortase B to understand differences in substrate specificity. These findings provide insights into the molecular mechanism of surface protein anchoring in Gram-positive bacteria.

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Area of Science:

  • Structural biology
  • Microbial pathogenesis
  • Protein-ligand interactions

Background:

Gram-positive bacteria like Staphylococcus aureus rely on surface proteins anchored to their cell walls to interact with host tissues. These proteins are covalently linked through a process involving a conserved LPXTG motif at their C-terminus. Sortase A (SrtA) is the enzyme responsible for cleaving this motif and anchoring proteins to the cell wall. Despite the importance of this process in bacterial pathogenesis, the structural details of SrtA and its interaction with substrates have remained unclear. Prior research has shown that SrtA cleaves the T-G bond in the LPXTG motif and forms an amide bond with cell wall components. However, the molecular architecture of the active site and the mechanism of substrate recognition have not been fully elucidated. This gap motivated the current study to determine the crystal structures of SrtA in both native and mutant forms, as well as in complex with its substrate. No prior work had resolved the spatial arrangement of conserved residues in the active site or the positioning of the scissile bond. Understanding these features is essential for grasping how Gram-positive bacteria anchor surface proteins to their cell walls.

Keywords:
sortase A functionGram-positive bacteriasurface protein anchoringX-ray crystallography

Frequently Asked Questions

The LPXTG motif is a conserved sequence at the C-terminus of surface proteins in S. aureus. Sortase A cleaves between the threonine and glycine residues of this motif to anchor proteins to the cell wall.

The active site mutant of SrtA retains the ability to bind the LPETG peptide but lacks catalytic activity. This allows researchers to study substrate recognition without cleavage.

The glutamic acid residue at the X position of the LPXTG motif extends into the solvent, which may allow for flexibility in substrate binding and positioning.

Structural comparisons show differences in the active site architecture of SrtA and sortase B, which may explain their distinct substrate specificities.

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Purpose Of The Study:

The purpose of this study was to determine the crystal structures of native and mutant forms of SrtA and its complex with the LPETG substrate peptide. The researchers aimed to reveal the structural basis of substrate recognition and catalysis by SrtA. By analyzing these structures, they sought to identify the residues involved in substrate binding and the positioning of the scissile T-G bond. The study also aimed to compare the active sites of SrtA and sortase B to understand differences in substrate specificity. The motivation for this work stems from the need to clarify the catalytic mechanism of SrtA, which is crucial for bacterial surface protein anchoring. The researchers proposed that understanding the structural features of SrtA could provide insights into the broader function of sortase enzymes in Gram-positive bacteria. This work addresses a key uncertainty in microbial pathogenesis related to the molecular mechanisms of surface protein anchoring.

Main Methods:

The study employed X-ray crystallography to determine the structures of native SrtA, an active site mutant of SrtA, and the mutant SrtA complexed with the LPETG peptide. The researchers crystallized these proteins and collected diffraction data to generate high-resolution structural models. They analyzed the structures to identify the residues involved in substrate binding and catalysis. The active site mutant was designed to disrupt catalytic activity and allow for substrate binding without cleavage. The LPETG peptide was used as a model substrate to mimic the natural substrate of SrtA. Structural comparisons were made between SrtA and sortase B to assess differences in active site architecture. The study also examined the positioning of conserved residues such as His(120), Cys(184), and Arg(197) in relation to the scissile T-G bond. These methods enabled the researchers to propose a model for the catalytic mechanism of SrtA.

Main Results:

The crystal structures revealed that the LPXTG motif residues are held in position by hydrophobic interactions, with the conserved proline and threonine residues forming stable contacts. The glutamic acid residue at the X position of the motif extends into the solvent, suggesting a role in substrate flexibility. The scissile T-G bond is positioned between the active site residues Cys(184) and Arg(197), with a greater distance from the imidazolium side chain of His(120). These three residues are conserved across Gram-positive sortase enzymes, indicating their importance in catalysis. The active site mutant of SrtA retained the ability to bind the LPETG peptide, confirming the role of these residues in substrate recognition. Structural comparisons between SrtA and sortase B showed differences in active site architecture that may explain substrate specificity. The study proposed a catalytic mechanism involving the positioning of the scissile bond between Cys(184) and Arg(197), with His(120) playing a supporting role. These findings provide a detailed structural framework for understanding the function of SrtA in surface protein anchoring.

Conclusions:

The study concludes that the active site of SrtA is composed of conserved residues that position the scissile T-G bond for catalysis. The hydrophobic interactions between the LPXTG motif and the enzyme are critical for substrate binding. The structural data suggest a catalytic mechanism involving Cys(184), Arg(197), and His(120), with the first two residues forming the active site and the latter playing a supporting role. The researchers propose that the conserved nature of these residues across Gram-positive sortases indicates a universal mechanism for surface protein anchoring. The comparison with sortase B highlights structural differences that may contribute to substrate specificity. The findings provide a molecular basis for understanding how SrtA functions in bacterial pathogenesis. The study does not suggest that these residues are essential for catalysis, but rather that they are conserved and likely important. The authors emphasize the need for further studies to confirm the proposed catalytic mechanism.

These residues are conserved across Gram-positive sortases and are positioned to facilitate the catalytic cleavage of the T-G bond in the LPXTG motif.

The study provides a structural framework for understanding how SrtA anchors surface proteins to the cell wall, which is essential for bacterial interaction with host tissues.