Abstract
Staphylococcus aureus is a dangerous human pathogen. A number of the proteins secreted by this bacterium are implicated in its virulence, but many of the components of its secretome are poorly characterized. Strains of S. aureus can produce up to six homologous extracellular serine proteases grouped in a single spl operon. Although the SplA, SplB, and SplC proteases have been thoroughly characterized, the properties of the other three enzymes have not yet been investigated. Here, we describe the biochemical and structural characteristics of the SplD protease. The active enzyme was produced in an Escherichia coli recombinant system and purified to homogeneity. P1 substrate specificity was determined using a combinatorial library of synthetic peptide substrates showing exclusive preference for threonine, serine, leucine, isoleucine, alanine, and valine. To further determine the specificity of SplD, we used high-throughput synthetic peptide and cell surface protein display methods. The results not only confirmed SplD preference for a P1 residue, but also provided insight into the specificity of individual primed- and non-primed substrate-binding subsites. The analyses revealed a surprisingly narrow specificity of the protease, which recognized five consecutive residues (P4-P3-P2-P1-P1’) with a consensus motif of R-(Y/W)-(P/L)-(T/L/I/V)↓S. To understand the molecular basis of the strict substrate specificity, we crystallized the enzyme in two different conditions, and refined the structures at resolutions of 1.56 Å and 2.1 Å. Molecular modeling and mutagenesis studies allowed us to define a consensus model of substrate binding, and illustrated the molecular mechanism of protease specificity.
Highlights
Staphylococcus aureus is a highly prevalent commensal bacterium that transiently or persistently colonizes the nares of 30%–70% of the human population without any detectable adverse effects [1]
The recombinant SplD protease was obtained by heterologous expression in E. coli as a glutathione S-transferase (GST) fusion protein
Chymotrypsin-like proteases are synthesized as inactive precursors containing Nterminal extensions
Summary
Staphylococcus aureus is a highly prevalent commensal bacterium that transiently or persistently colonizes the nares of 30%–70% of the human population without any detectable adverse effects [1]. The proteases, as a group, are of great importance to the virulence of the bacterium [7,8]. Individual proteases have diverging roles in the infection process, including inactivation of the host’s protease inhibitors and antimicrobial peptides, modulation of kinin and chemokine synthesis, degradation of immunoglobulins and complement cascade proteins, modification of the bacterial surface, interactions with components of the coagulation and fibrinolysis pathways, and other [9,10,11,12,13,14,15]. The specific contributions of the staphylococcal proteolytic system and its individual proteases, except for epidermolytic toxins, to the pathogenicity of S. aureus in vivo are still far from being fully understood
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