title: 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.
authors.affiliation: Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
authors.forename: Michal
authors.initials: M
authors.lastname: Zdzalik
authors.forename: Magdalena
authors.initials: M
authors.lastname: Kalinska
authors.forename: Magdalena
authors.initials: M
authors.lastname: Wysocka
authors.forename: Justyna
authors.initials: J
authors.lastname: Stec-Niemczyk
authors.forename: Przemyslaw
authors.initials: P
authors.lastname: Cichon
authors.forename: Natalia
authors.initials: N
authors.lastname: Stach
authors.forename: Natalia
authors.initials: N
authors.lastname: Gruba
authors.forename: Henning R
authors.initials: HR
authors.lastname: Stennicke
authors.forename: Abeer
authors.initials: A
authors.lastname: Jabaiah
authors.forename: Michal
authors.initials: M
authors.lastname: Markiewicz
authors.forename: Sylwia
authors.initials: S
authors.lastname: Kedracka-Krok
authors.forename: Benedykt
authors.initials: B
authors.lastname: Wladyka
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Daugherty
authors.forename: Adam
authors.initials: A
authors.lastname: Lesner
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Rolka
authors.forename: Adam
authors.initials: A
authors.lastname: Dubin
authors.forename: Jan
authors.initials: J
authors.lastname: Potempa
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Dubin
mesh_terms: D020134:Catalytic Domain; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D059748:Proteolysis; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D010455:Peptides; D010447:Peptide Hydrolases