Table 1.
Data collection, structure determination and refinement statistics.
Figure 1.
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.
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.
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.
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.
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.
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.