Biochemical and Structural Characterization of SplD Protease from Staphylococcus aureus

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.

Mapping the Global Network of Extracellular Protease Regulation inStaphylococcus aureus

“…Specifically, MgrA, SarR and SaeR each bring about profound upregulation of the spl operon, to levels that rival and, in the case of SaeR, exceed that of SarA and CodY for protease control. This is of interest because the Spls are well known for their narrow substrate specificity [58–60]. Indeed, these enzymes share strong homology and many enzymatic characteristics with the exfoliative toxins of S. aureus .…”

Staphylococcus Aureus V8 protease disrupts the integrity of the airway epithelial barrier and impairs IL‐6 production in vitro

“…ETA was expressed in E. coli and purified, as earlier described . SplA, SplB, SplC, and SplD were expressed in E. coli , as described previously . SplE and SplF were purified as detailed in the supporting information (see supporting document: Supplementary Methods).…”

“…24,25 SplA, SplB, SplC, and SplD were expressed in E. coli, as described previously. [26][27][28][29] SplE and SplF were purified as detailed in the supporting information (see supporting document: Supplementary Methods). The purity of all preparations was at least 95%, as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.…”

Novel roles of staphylococcal proteases and cross talk in biofilm formation and virulence

“…These are W/Y-L-Y-S/T for SplA [235], W-E-L-Q for SplB [233], and R-Y/W-P/L-T/L/I/V-S for SplD [236]. We therefore cannot predict any specific hits that are directly cleaved.…”

“…The Spl proteases are an intriguing area of study because, despite evidence that they contribute to S. aureus-host interaction, their specific functions have not been identified. Structural and activity analyses of SplA, SplB, and SplD have revealed that these enzymes are likely highly specific, given that they have cleavage preferences in subsites beyond the P1 residue of cleavage [232,233,235,236]. Further, the Spls are not produced as zymogens, but in some cases, appear to require binding of a preferred substrate in order to take on an enzymatically active conformation [232], as has been demonstrated by activity characterization of SplB [233].…”