Interrogation of 3D-swapped structure and functional attributes of quintessential Sortase A from Streptococcus pneumoniae.

Tora Biswas,Suryanarayanarao Ramakumar,Rajendra Roy,Prity Yadav,Sreetama Das,Anurag Misra

doi:10.1042/bcj20200631

Abstract

The anchoring of the surface proteins to the cell wall in gram-positive bacteria involves a peptide ligation reaction catalyzed by transpeptidase sortase. Most bacterial genomes encode multiple sortases with dedicated functions. Streptococcus pneumoniae (Sp) carries four sortases; a housekeeping sortase (SrtA), and three pilin specific sortases (SrtC1, C2, C3) dedicated to the biosynthesis of covalent pilus. Interestingly, SrtA, meant for performing housekeeping roles, is also implicated in pilus assembly of Sp. The allegiance of SpSrtA to the pathogenic pilus assembly makes it an ideal target for clinical inhibitor development. In this paper, we describe biochemical characterization, crystal structure and peptide substrate preference of SpSrtA. Transpeptidation reaction with a variety of substrates revealed that the enzyme preferred elongated LPXTG sequences and transferred them equally well to both Ala- and Gly-terminated peptides. Curiously, crystal structure of both wild type and an active site (Cys to Ala) mutant of SpSrtA displayed inter-twined 3D-swapped dimers in which each protomer generated a classic eight stranded beta-barrel "sortase fold". Size-exclusion chromatography and sedimentation equilibrium measurements revealed predominant presence of a dimer in equilibrium with its monomer. The crystal structure-based Cys-Cys distance mapping with defined chemical cross-linkers established the existence of 3D-swapped structure in solution. The swapping in SpSrtA, unprecedented for sortase family, may be physiologically relevant and meant to perform regulatory functions.

Highlights

Streptococcus pneumoniae (S. pneumoniae) is a gram-positive bacterium and a commensal of human nasopharyngeal cavity
Sortase enzymes are implicated in bacterial pathogenesis, and serve as effective protein labelling tool [8]
Structure and specificity elucidation as a prelude to developing clinical inhibitors or improved sortases for protein engineering applications remains a topical subject in sortase research

Summary

Introduction

Streptococcus pneumoniae (S. pneumoniae) is a gram-positive bacterium and a commensal of human nasopharyngeal cavity. The sortase-mediated covalent anchoring of proteins proceeds through a thioacyl-enzyme intermediate generated by nucleophilic attack of the thiol group of the catalytic Cys residue on the scissile T-G peptide bond of the LPXTG motif [10]. Subsequent reaction of the thioacyl-enzyme intermediate with the terminal amine group of the pentaglycine/dialanine branch of the peptidoglycan results in the formation of a peptide bond leading to the covalent attachment of the surface protein to the peptidoglycan. Sortases involved in pilin assembly use epsilon amine of a specific Lys residue present in the pilin subunit to resolve the thioacyl-enzyme intermediate and link the pilin chains by an isopeptide bond [9]

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Conclusion