Abstract
The assembly of pili on the cell wall of Gram-positive bacteria requires transpeptidase enzymes called sortases. In Streptococcus agalactiae, the PI-1 pilus island of strain 2603V/R encodes two pilus-specific sortases (SrtC1 and SrtC2) and three pilins (GBS80, GBS52 and GBS104). Although either pilus-specific sortase is sufficient for the polymerization of the major pilin, GBS80, incorporation of the minor pilins GBS52 and GBS104 into the pilus structure requires SrtC1 and SrtC2, respectively. The S. agalactiae housekeeping sortase, SrtA, whose gene is present at a different location and does not catalyze pilus polymerization, was shown to be involved in cell wall anchoring of pilus polymers. To understand the structural basis of sortases involved in such diverse functions, we determined the crystal structures of S. agalactiae SrtC1 and SrtA. Both enzymes are made of an eight-stranded beta-barrel core with variations in their active site architecture. SrtA exhibits a catalytic triad arrangement similar to that in Streptococcus pyogenes SrtA but different from that in Staphylococcus aureus SrtA. In contrast, the SrtC1 enzyme contains an N-terminal helical domain and a ‘lid’ in its putative active site, which is similar to that seen in Streptococcus pneumoniae pilus-specific sortases, although with subtle differences in positioning and composition. To understand the effect of such differences on substrate recognition, we have also determined the crystal structure of a SrtC1 mutant, in which the conserved DP(W/F/Y) motif was replaced with the sorting signal motif of GBS80, IPNTG. By comparing the structures of WT wild type SrtA and SrtC1 and the ‘lid’ mutant of SrtC1, we propose that structural elements within the active site and the lid may be important for defining the role of specific sortase in pili biogenesis.
Highlights
In Gram-positive bacteria, cell wall-anchored surface proteins are virulence factors and are essential for host-cell adhesion, nutrient acquisition and many other functions that facilitate pathogen survival in a hostile environment [1,2]
Based on the S. aureus sortase A (SASrtA)-substrate complex structure [40], we suggest an elongated cleft that widens towards the catalytic Cys184 residue on one end is the putative active site of GBSSrtC1, with the b7, b8 and b4 strands as its floor and the b6/ b7, b3/b4 and the b2/helix5 loops as the walls of this binding pocket (Figure 2b and 3b)
The latter step involves the insertion of a minor pilin at the pilus base, which is anchored to the cell wall and for both these steps the housekeeping sortase is essential
Summary
In Gram-positive bacteria, cell wall-anchored surface proteins are virulence factors and are essential for host-cell adhesion, nutrient acquisition and many other functions that facilitate pathogen survival in a hostile environment [1,2]. Anchoring of these surface proteins to the cell wall peptidoglycan requires transpeptidase enzymes called sortases, a product of an srt (surface protein sorting) gene [3,4]. The substrates of sortases are protein precursors with an N-terminal signal peptide and a C-terminal cell wall sorting signal (CWSS), consisting of the LPXTG motif, followed by a segment of hydrophobic and basic residues [1]. The subsequent transglycosylation and transpeptidation reactions incorporate the surface protein into the peptidoglycan and decorate the bacterial cell surface with covalently bound proteins [6]
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