1. Sortase-Mediated Pilus Fiber Biogenesis in Streptococcus pneumoniae
Clothilde Manzano, Carlos Contreras-Martel, Lamya El Mortaji, Thierry Izoré, Daphna Fenel, Thierry Vernet, Guy Schoehn, Anne Marie Di Guilmi, Andréa Dessen 
Structure 
Volume 16, Issue 12, Pages (December 2008)
DOI: /j.str
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2. Figure 1 SrtC-1 Is a Pilus-Polymerizing Transpeptidase
(A) Western blots using polyacrylamide gradient gels developed with RrgB antiserum. Lanes: (1), monomeric RrgB; (2), RrgB + SrtC-1; (3), RrgB + SrtC-2; (4), RrgB + SrtC-3; (5) RrgB + SrtC-1Cys193Ala; (6) total extract from mutanolysed TIGR4; (7) supernatant from mutanolysed TIGR4; (8) total extract from mutanolysed TIGR4ΔSP0466; (9) supernatant from mutanolysed TIGR4ΔSP0466. Mutanolysed samples display pili that harbor not only polymerized RrgB, but also associated RrgA and RrgC, as well as peptidoglycan fragments. (10) Gel-filtered sample analyzed by electron microscopy and LCMS/MS.
(B) Top, negative staining electron microscopy images of the RrgB fibers generated by SrtC-1 activity. Black arrows point to fibers whose diameters are in the range of 3.5 nm or 7.0 nm. Bottom, a gallery of typical fibers seen on grids. Scale bar, 20 × 5 nm.
Structure  , DOI: ( /j.str )
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3. Figure 2
Sortases of the Pilus-Formation Machinery Display Similar Folds
(A and B) SrtC-1 (A) and SrtC-3 (B) fold into β-barrels surrounded by helices; their active sites, centered on the region surrounding the C terminus of β7 and the N terminus of β8, are covered by a lid (shown in pink).
(C) Structure-based sequence alignment of the three pilus-forming sortases of S. pneumoniae. Identical residues are shown with a red background, whereas similar residues are shown in red and highlighted with blue boxes. Residues located within the active site cleft of SrtC-1/SrtC-3 are highlighted in green, whereas the Asn residue on β7/β8 that contacts the nucleophilic Cys in SrtC-1 is shown with an orange background. The lid region is shown in magenta. The TM (transmembrane) region was predicted with the SMART server (
Structure  , DOI: ( /j.str )
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4. Figure 3
The Active Site of Pilus-Forming Sortases Is Anchored by a Lid
(A) The nucleophilic Cys 193 in SrtC-1 displays two conformations, but only one is shown for clarity.
(B) The active site of SrtC-3, like SrtC-1, displays a catalytic Arg residue whose side chain is stably anchored by an Asp residue on the lid “anchor” region. In both active sites, the conserved His residue is approximately 5.4 Å away from the Cys nucleophile.
(C) The active site of SrtA from S. aureus (Protein Data Bank code 1T2P) is mostly solvent-exposed and does not harbor a lid.
Structure  , DOI: ( /j.str )
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5. Figure 4 The Substrate-Binding Cleft of Sortases Is Conserved
(A) Surface of SrtC-1, onto which residues that are conserved (orange) and identical (red) among the three pilus-forming sortases and have been mapped. The analogous residues were mapped onto the surface of S. aureus SrtA (B). This analysis identifies that the lid of pilus-forming sortases (shown in green) is positioned analogously to the LPETG substrate peptide (shown in cyan) in the structure of SrtA, suggesting that the substrate-recognition cleft in pilus-forming sortases is sterically blocked by the lid. Active site residues are shown in dark blue.
Structure  , DOI: ( /j.str )
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6. Figure 5
Surface Potential Representations of All Three Pilus-Forming Sortases, with a Direct View onto the Active Site
The structure of SrtC-2 was generated by building a model based on the structure of SrtC-1, with which it shares 55.7% sequence identity. Striking features include the highly basic character of the SrtC-1 cleft and the vast acidic region in the vicinity of the SrtC-3 active site (same orientation as in Figure 3).
Structure  , DOI: ( /j.str )
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