Structure of the mature Streptococcal cysteine protease exotoxin mSpeB in its active dimeric form.
ABSTRACT Invasive infections of Streptococcus pyogenes are dependent on the cysteine protease streptococcal pyrogenic exotoxin B. Previous structures of the enzyme have not disclosed the proper active-site configuration. Here, the crystal structure of the mature enzyme is presented to 1.55 A, disclosing a homodimer. A serine from one subunit inserts into the active site of the other to donate to the oxyanion hole and coordinates the ligand proximal to the active-site cysteine. Dimerization is unique to the mature form and is clearly a prerequisite for catalysis. The present structure supports a tripartite switch system that is triggered upon dimerization and substrate binding: (1) liberation of the active-site histidine from an inactive configuration, (2) relocation of residues blocking the substrate binding pockets and (3) repositioning of two active-site tryptophans to settle in the active configuration. Based on the present structure, the active site of clan CA cysteine proteases is expanded and a detailed mechanism of the deacylation mechanism is proposed. The results may have applications for the development of protease inhibitors specific to bacterial cysteine proteases.
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ABSTRACT: Group A streptococcus (Streptococcus pyogenes) is an exclusively human pathogen that causes a wide spectrum of diseases ranging from pharyngitis, to impetigo, to toxic shock, to necrotizing fasciitis. The diversity of these disease states necessitates that S. pyogenes possess the ability to modulate both the innate and adaptive immune responses. SpeB, a cysteine proteinase, is the predominant secreted protein from S. pyogenes. Because of its relatively indiscriminant specificity, this enzyme has been shown to degrade the extracellular matrix, cytokines, chemokines, complement components, immunoglobulins, and serum protease inhibitors, to name but a few of the known substrates. Additionally, SpeB regulates other streptococcal proteins by degrading them or releasing them from the bacterial surface. Despite the wealth of literature on putative SpeB functions, there remains much controversy about this enzyme because many of reported activities would produce contradictory physiological results. Here we review all known host and bacterial protein substrates for SpeB, their cleavage sites, and discuss the role of this enzyme in streptococcal pathogenesis based on the current literature.Biological Chemistry 12/2011; 392(12):1077-88. · 2.68 Impact Factor
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ABSTRACT: Streptococcal pyrogenic exotoxin B (SpeB) is a protease secreted by group A streptococci and known to degrade a wide range of host and GAS proteins in vitro. Although the role of SpeB in GAS infection is debated, recent evidence has conclusively demonstrated that SpeB is critical for the pathogenesis of severe invasive disease caused by GAS. Genetic inactivation of the speB gene results in significantly decreased virulence in a necrotizing fasciitis model of infection. Production of fully active SpeB by GAS is extremely complex. Following transcription and translation the SpeB protein is secreted as an inactive zymogen, which is autocatalytically processed through a series of intermediates to form an active protease. Each step from transcription to protease activation is tightly controlled and regulated by the bacterial cell reflecting the critical role played by this virulence factor in GAS infection. Here we review the molecular aspects of SpeB production by GAS from transcription to activation and the multiple layers of control involved.Molecular Microbiology 06/2011; 81(3):588-601. · 5.03 Impact Factor
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ABSTRACT: Cysteine protease SpeB is secreted from Streptococcus pyogenes and has been studied as a potential virulence factor since its identification almost 70 years ago. Here, we report the crystal structures of apo mature SpeB to 1.06 Å resolution as well as complexes with the general cysteine protease inhibitor trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane and a novel substrate mimetic peptide inhibitor. These structures uncover conformational changes associated with maturation of SpeB from the inactive zymogen to its active form and identify the residues required for substrate binding. With the use of a newly developed fluorogenic tripeptide substrate to measure SpeB activity, we determined IC(50) values for trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane and our new peptide inhibitor and the effects of mutations within the C-terminal active site loop. The structures and mutational analysis suggest that the conformational movements of the glycine-rich C-terminal loop are important for the recognition and recruitment of biological substrates and release of hydrolyzed products.Journal of Biological Chemistry 05/2012; 287(29):24412-26. · 4.65 Impact Factor