Sortase-Mediated Pilus Fiber Biogenesis in Streptococcus pneumoniae

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369

In Gram-positive bacteria, sortase enzymes assemble surface proteins and pili in the cell wall envelope. Sortases catalyze a transpeptidation reaction that joins a highly conserved LPXTG sorting signal within their polypeptide substrate to the cell wall or to other pilin subunits. The molecular basis of transpeptidation and sorting signal recognition are not well understood, because the intermediates of catalysis are short lived. We have overcome this problem by synthesizing an analog of the LPXTG signal whose stable covalent complex with the enzyme mimics a key thioacyl catalytic intermediate. Here we report the solution structure and dynamics of its covalent complex with the Staphylococcus aureus SrtA sortase. In marked contrast to a previously reported crystal structure, we show that SrtA adaptively recognizes the LPXTG sorting signal by closing and immobilizing an active site loop. We have also used chemical shift mapping experiments to localize the binding site for the triglycine portion of lipid II, the second substrate to which surface proteins are attached. We propose a unified model of the transpeptidation reaction that explains the functions of key active site residues. Since the sortase-catalyzed anchoring reaction is required for the virulence of a number of bacterial pathogens, the results presented here may facilitate the development of new anti-infective agents.Bacterial surface proteins function as virulence factors that enable pathogens to adhere to sites of infection, evade the immune response, acquire essential nutrients, and enter host cells (1). Gram-positive bacteria use a common mechanism to covalently attach proteins to the cell wall. This process is catalyzed by sortase transpeptidase enzymes, which join proteins bearing a highly conserved Leu-Pro-X-Thr-Gly (LPXTG, where X is any amino acid) sorting signal to the cross-bridge peptide of the peptidylglycan (2-4). Sortases also polymerize proteins containing sorting signals into pili, filamentous surface exposed structures that promote bacterial adhesion (5, 6). The search for small molecule sortase inhibitors is an active area of research, since these enzymes contribute to the virulence of a number of important pathogens, including among others Staphylococcus aureus, Listeria monocytogenes, Streptococcus pyogenes, and Streptococcus pneumoniae (reviewed in Refs. 7 and 8). Sortase enzymes are also promising molecular biology reagents that can be used to site-specifically attach proteins to a variety of biomolecules (9 -14, 72).The sortase A (SrtA) 7 enzyme from S. aureus is the prototypical member of the sortase enzyme family (15, 16). It anchors proteins to the murein sacculus that possess a COOH-terminal cell wall sorting signal that consists of a LPXTG motif, followed by a hydrophobic segment of amino acids and a tail composed of mostly positively charged residues (17). SrtA is located on the extracellular side of the membrane. After partial secretion of its protein substrate across the cell membrane, SrtA cleaves the LPXTG motif between...

Section: Some Sortase Enzymes May Contain a Preformed Binding Pocket mentioning

confidence: 90%

Section: Some Sortase Enzymes May Contain a Preformed Binding Pocket mentioning

confidence: 99%

mentioning

confidence: 99%

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The Structure of the Staphylococcus aureus Sortase-Substrate Complex Reveals How the Universally Conserved LPXTG Sorting Signal Is Recognized

Suree¹,

Liew²,

Villareal³

et al. 2009

172

369

“…These membrane-associated sortase enzymes recognize a LPXTG-like sequence (sorting motif) within their protein substrate (11,12). The enzymatic reaction involves a nucleophilic cysteine residue, which is essential for the covalent intermolecular association between pilus subunits (13)(14)(15). In addition, intramolecular bonds within pilin subunits, formed between lysine and asparagine side chains, have been identified in the high resolution structures of major pilins Spy0128 from S. pyogenes (16) and SpaA from C. diphtheriae (17) as well as in the main adhesin of the Streptococcus pneumoniae pilus, RrgA (18).…”

mentioning

confidence: 99%

Stability and Assembly of Pilus Subunits of Streptococcus pneumoniae

Mortaji

Terrasse

Dessen

et al. 2010

Self Cite

Pili are surface-exposed virulence factors involved in bacterial adhesion to host cells. The Streptococcus pneumoniae pilus is composed of three structural proteins, RrgA, RrgB, and RrgC and three transpeptidase enzymes, sortases SrtC-1, SrtC-2, and SrtC-3. To gain insights into the mechanism of pilus formation we have exploited biochemical approaches using recombinant proteins expressed in Escherichia coli. Using site-directed mutagenesis, mass spectrometry, limited proteolysis, and thermal stability measurements, we have identified isopeptide bonds in RrgB and RrgC and demonstrate their role in protein stabilization. Co-expression in E. coli of RrgB together with RrgC and SrtC-1 leads to the formation of a covalent RrgB-RrgC complex. Inactivation of SrtC-1 by mutation of the active site cysteine impairs RrgB-RrgC complex formation, indicating that the association between RrgB and RrgC is specifically catalyzed by SrtC-1. Mass spectrometry analyses performed on purified samples of the RrgB-RrgC complex show that the complex has 1:1 stoichiometry. The deletion of the IPQTG RrgB sorting signal, but not the corresponding sequence in RrgC, abolishes complex formation, indicating that SrtC-1 recognizes exclusively the sorting motif of RrgB. Finally, we show that the intramolecular bonds that stabilize RrgB may play a role in its efficient recognition by SrtC-1. The development of a methodology to generate covalent pilin complexes in vitro, facilitating the study of sortase specificity and the importance of isopeptide bond formation for pilus biogenesis, provide key information toward the understanding of this complex macromolecular process.

“…The backbone is formed by the covalent association of RrgB monomers (Fig. 6, bright green), and its nucleophilic Lys-183 residue thus has a dual recognition potential, participating in the covalent association with RrgA-YPRTG and to the IPQTG motif of other RrgB molecules to generate the fiber with the adhesin at its tip (21,24,25,59). Finally, RrgC (Fig.…”

Section: Discussionmentioning

confidence: 99%

Structural Basis of Pilus Anchoring by the Ancillary Pilin RrgC of Streptococcus pneumoniae

Shaik

Maccagni

Tourcier

et al. 2014

Self Cite

Background: RrgC is a key component of the pneumococcal pilus, a virulence factor that plays an important role in pathogenesis. Results: RrgC folds into three independent domains and requires the housekeeping sortase for surface association. Conclusion: The rod-like structure of RrgC suggests that it stably bridges peptidoglycan and pilus fiber. Significance: A complete model of the pneumococcal pilus reveals a multidomain, flexible assembly.