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, 6 (1), e15969

Crystal Structure of Spy0129, a Streptococcus Pyogenes Class B Sortase Involved in Pilus Assembly


Crystal Structure of Spy0129, a Streptococcus Pyogenes Class B Sortase Involved in Pilus Assembly

Hae Joo Kang et al. PLoS One.


Sortase enzymes are cysteine transpeptidases that mediate the covalent attachment of substrate proteins to the cell walls of gram-positive bacteria, and thereby play a crucial role in virulence, infection and colonisation by pathogens. Many cell-surface proteins are anchored by the housekeeping sortase SrtA but other more specialised sortases exist that attach sub-sets of proteins or function in pilus assembly. The sortase Spy0129, or SrtC1, from the M1 SF370 strain of Streptococcus pyogenes is responsible for generating the covalent linkages between the pilin subunits in the pili of this organism. The crystal structure of Spy0129 has been determined at 2.3 Å resolution (R = 20.4%, Rfree = 26.0%). The structure shows that Spy0129 is a class B sortase, in contrast to other characterised pilin polymerases, which belong to class C. Spy0129 lacks a flap believed to function in substrate recognition in class C enzymes and instead has an elaborated β6/β7 loop. The two independent Spy0129 molecules in the crystal show differences in the positions and orientations of the catalytic Cys and His residues, Cys221 and His126, correlated with movements of the β7/β8 and β4/β5 loops that respectively follow these residues. Bound zinc ions stabilise these alternative conformations in the crystal. This conformational variability is likely to be important for function although there is no evidence that zinc is involved in vivo.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Structure of Spy0129.
A) Stereo view of the monomer (molecule B), colour coded from N-terminus (blue) to C-terminus (red). The putative catalytic residues Cys221 and His126 are shown in stick representation. B) Topology diagram for Spy0129, coloured as in A) and labelled from α1 to α6 for α-helices and β1 to β8 for β-strands.
Figure 2
Figure 2. Sequence alignment of Spy0129 with other class B sortases.
Invariant residues are highlighted in dark blue and conserved residues in lighter blue colours. Putative catalytic residues are coloured in purple. The secondary structure elements from Spy0129 are shown above the sequence. Residues corresponding to the sortase signature motif are boxed with a black outline. The β6/β7 loop region of S. aureus SrtB which was shown to confer substrate specificity is outlined with a dotted red box.
Figure 3
Figure 3. Comparison of different sortase structures.
A) Class B: Spy0129 from S. pyogenes (this work); B) Class B: SrtB from S. aureus (PDB code 1T2P): C) Class C: SrtC1 from S. pneumoniae (PDB code 2W1J); and D) Class A: SrtA-LPAT* complex structure from S.aureus (PDB code 2KID). In each case, the β6/β7 region is highlighted with darker colour than the rest of the molecule. The lockable lid in the pneumococcal SrtC1 is highlighted in blue. All four structures are shown in equivalent orientations. The catalytic Cys residues are shown in stick mode.
Figure 4
Figure 4. Active site of Spy0129.
Stereo view of the active site region, with molecule A (magenta) superimposed on to molecule B (light teal) to show the conformational differences in the β4/β5 and β7/β8 loops. The residues Cys221, His126, His127 and Arg229 are shown in stick mode, coloured to correspond to the molecule to which they belong. In molecule A, Cys221 is linked to His127 through a bound zinc ion (magenta sphere) and His126 is oriented away from Cys221. In molecule B, Cys221 is linked to His126 through a zinc atom (blue sphere) and His127 is oriented away.
Figure 5
Figure 5. Surface structures involved in substrate binding.
Stereo diagram of Spy0129 (magenta), with its β6/β7 region highlighted in darker magenta. Superimposed on to Spy0129 are the lockable lid present in the class C SrtC enzymes from S. pneumoniae (dark blue) and β6/β7 region from S. aureus SrtA (dark gray) including the 310-helix that helps bind the LPAT* peptide analogue.
Figure 6
Figure 6. In vitro polymerisation assay.
A) Immunoblot with polyclonal antibodies against Spy0128, after mixing Spy0128 and Spy0129 and incubating at 37°C for 20 h, showing the lack of a ladder of high molecular weight bands characteristic of polymer formation. B) Immunoblot of S. pyogenes M1 SF370 pili, composed mainly of polymerised Spy0128, probed with Spy0128 antibodies.

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    1. Mazmanian SK, Liu G, Ton-That H, Schneewind O. Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science. 1999;285:760–763. - PubMed
    1. Schneewind O, Model P, Fischetti VA. Sorting of protein A to the staphylococcal cell wall. Cell. 1992;70:267–281. - PubMed
    1. Jonsson IM, Mazmanian SK, Schneewind O, Verdrengh M, Bremell T, et al. On the role of Staphylococcus aureus sortase and sortase-catalyzed surface protein anchoring in murine septic arthritis. J Infect Dis. 2002;185:1417–1424. - PubMed
    1. Kharat AS, Tomasz A. Inactivation of the srtA gene affects localization of surface proteins and decreases adhesion of Streptococcus pneumoniae to human pharyngeal cells in vitro. Infect Immun. 2003;71:2758–2765. - PMC - PubMed
    1. Mazmanian SK, Liu G, Jensen ER, Lenoy E, Schneewind O. Staphylococcus aureus sortase mutants defective in the display of surface proteins and in the pathogenesis of animal infections. Proc Natl Acad Sci U S A. 2000;97:5510–5515. - PMC - PubMed

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