Two Crystal Structures of Pneumococcal Pilus Sortase C Provide Novel Insights into Catalysis and Substrate Specificity

The pathogen Bacillus anthracis uses the Sortase A (SrtA) enzyme to anchor proteins to its cell wall envelope during vegetative growth. To gain insight into the mechanism of protein attachment to the cell wall in B. anthracis we investigated the structure, backbone dynamics, and function of SrtA. The NMR structure of SrtA has been determined with a backbone coordinate precision of 0.40 ؎ 0.07 Å . SrtA possesses several novel features not previously observed in sortase enzymes including the presence of a structurally ordered amino terminus positioned within the active site and in contact with catalytically essential histidine residue (His 126 ). We propose that this appendage, in combination with a unique flexible active site loop, mediates the recognition of lipid II, the second substrate to which proteins are attached during the anchoring reaction. pK a measurements indicate that His 126 is uncharged at physiological pH compatible with the enzyme operating through a "reverse protonation" mechanism. Interestingly, NMR relaxation measurements and the results of a model building study suggest that SrtA recognizes the LPXTG sorting signal through a lock-in-key mechanism in contrast to the prototypical SrtA enzyme from Staphylococcus aureus.

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The Sortase A Enzyme That Attaches Proteins to the Cell Wall of Bacillus anthracis Contains an Unusual Active Site Architecture

Weiner

Robson

Marohn

et al. 2010

“…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).…”

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confidence: 99%

Stability and Assembly of Pilus Subunits of Streptococcus pneumoniae

Mortaji

Terrasse

Dessen

et al. 2010

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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.

“…We then set out to investigate the transpeptidation roles of SrtC-1 and SrtC-3, which had been previously linked to RrgC functionality within the pilus (19,23,52). To do so, we expressed different combinations of pilus-related proteins in S. pneumoniae R6 and R6⌬srtA strains and tested for the presence of RrgC in total, cell wall, and membrane extracts (Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Shaik

Maccagni

Tourcier

et al. 2014

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.