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

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.

Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes

“…12 Because spSrtA crystallizes as a domain-swapped dimer, which is enzymatically inactive in our hands, we used previously published Class A sortase structures to investigate the stereochemistry of our biochemical results. 12,40,41 Now, we investigate two additional sortases, those from S. pyogenes (spySrtA) and S. agalactiae (sagSrtA), to see if the β7-β8 loop has broad effects on enzyme function and target recognition for Streptococcus Class A sortases.…”

Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes

“…Both spySrtA and sagSrtA 238 were previously crystallized and we reasoned that experimental structural data would enable an understanding of the stereochemistry of sortase-substrate interactions in a way not available to spSrtA, which crystallizes as a catalytically inactive domain-swapped dimer. 40,41,43,45…”

“…In addition, the only available spSrtA structures in the Protein Data Bank are of domain-swapped dimers, which are not enzymatically active in our hands (data not shown). 40,41 Considering the observations about contributions of individual residues to activity and/or selectivity by ourselves and others, there remains much to be learned from the study of individual sortase enzymes.…”

“…Both spySrtA and sagSrtA238 were previously crystallized and we reasoned that experimental structural data would enable an understanding of the stereochemistry of sortase-substrate interactions in a way not available to spSrtA, which crystallizes as a catalytically inactive domain-swapped dimer. 40,41,43,45 Beginning with sagSrtA, an important consideration for the reported sagSrtA238 structure was that we found this variant to be inactive in our enzyme assays (Figure 2b). The crystal structure of sagSrtA238 shows a dodecameric protein comprised of two hexameric rings (Figure S4a).…”

“…12 Because spSrtA crystallizes as a domain-swapped dimer, which is enzymatically inactive in our hands, we used previously published Class A sortase structures to investigate the stereochemistry of our biochemical results. 12,40,41 Now, we investigate two additional sortases, those from S. pyogenes (spySrtA) and S. agalactiae (sagSrtA), to see if the b7-b8 loop has broad effects on enzyme function and target recognition for Streptococcus Class A sortases.…”