Streptococcus pyogenesSpyCEP: a chemokine‐inactivating protease with unique structural and biochemical features

Abstract

SpyCEP is a 170-kDa multidomain serine protease expressed on the surface of the human pathogen Streptococcus pyogenes, which plays an important role in infection by catalyzing cleavage and inactivation of the neutrophil chemoattractant interleukin-8. In this study, we investigated the biochemical features and maturation process of SpyCEP, starting from a recombinant form of the protease expressed and purified from Escherichia coli. We show that active recombinant SpyCEP differs from other bacterial proteases in that it is constituted by 2 noncovalently linked fragments derived from autocatalytic processing, an N-terminal fragment of 210 aa bearing one of the 3 catalytic triad residues, and a 1369-residue C-terminal polypeptide containing the remaining 2 catalytic amino acids. The same type of organization is present in the enzyme obtained from S. pyogenes. Furthermore, N-terminal SpyCEP is not involved in the folding of the mature enzyme. The 2 protease fragments were separately expressed in E. coli as soluble polypeptides that, when combined, reconstituted a fully active enzyme complex. Therefore, SpyCEP appears to possess a completely new structural architecture that has not been described so far for other microbial proteases.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of SpyCEP, a candidate antigen for a vaccine againstStreptococcus pyogenes

“…The stability of SpyCEP , and the relative instability of the SpyCEP 113-244 domain, have been described previously (Zingaretti et al, 2010). Here, a new construct, SpyCEP 245-1131 , lacking $500 Cterminal residues, was prepared.…”

“…Using PIPE PCR cloning (Klock & Lesley, 2009) and the GAS M1 strain SpyCEP ectodomain (residues 34-1613; UniProt Q9A180) in pET-21b (Zingaretti et al, 2010), three SpyCEP constructs were prepared in pET-28a, termed SpyCEP 113-244 , SpyCEP 245-1131 and SpyCEP (Fig. 1a), excluding residues 33-112 because of predicted disorder and excluding residues 1132 onwards in order to mimic the C5a peptidase construct used for structure determination (Kagawa et al, 2009).…”

“…1a), excluding residues 33-112 because of predicted disorder and excluding residues 1132 onwards in order to mimic the C5a peptidase construct used for structure determination (Kagawa et al, 2009). The catalytic residues Asp151 and Ser617 were mutated to Ala, as described previously (Zingaretti et al, 2010), to minimize proteolysis during crystallization. Each construct was cloned with an additional Met at the N-terminus, while at the Cterminus each construct included a hexapeptide linker (sequence GSALAE) added between the SpyCEP residues and the His 6 tag to facilitate access to the C-terminal His 6 tag.…”

“…SpyCEP is immunogenic in animals and humans and, either alone or in a combination of three GAS proteins, SpyCEP confers protection in animals against multiple GAS serotypes ( Lei et al, 2000;Rodríguez-Ortega et al, 2006;Fritzer et al, 2009;Zingaretti et al, 2010;Bensi et al, 2012).…”

“…SpyCEP contains distinct N-terminal and C-terminal domains separated by autocleavage between residues Gln244 and Ser245. Interestingly, both domains contribute residues to the catalytic triad (Asp151, His279 and Ser617) and can be produced separately for subsequent reconstitution of the functional enzymatic complex (Fritzer et al, 2009;Zingaretti et al, 2010). The three-dimensional structure of SpyCEP is currently unknown; the closest known homologous structure is likely to be the S. pyogenes C5a peptidase (UniProt P15926; PDB entry 3eif; Kagawa et al, 2009), which shares 32% sequence identity with SpyCEP residues Ser118-Leu1128.…”