Chapter 7 : Sortase Pathways in Gram-Positive Bacteria

Mechanisms of cell wall attachment. Choline-binding proteins recognize choline-substituted TA and LTA (asterisks) through 20-residue C-terminal repeats, which form stacked β-hairpins. LTA can be directly bound by the GW domains of InlB. Crystalline S-layer proteins self-assemble at the cell surface; they are tethered to TUA polymers through SLH domains. The SLH domain of ScaC affixes it to the membrane, and its three cohesin domains serve as attachment points for the adaptor proteins and enzymes of the cellulosome. Covalent linkage of Cws-containing proteins by a sortase enzyme produces an amide linkage between the threonine of the LPXTG motif of the sorting signal and the peptide cell wall crossbridge. Adapted from Cossart and Jonquieres (2000) .

(A) Schematic of a sortase substrate. The protein is composed of an N-terminal signal peptide and a C-terminal Cws. The Cws contains a conserved LPXTG motif followed by a hydrophobic stretch of amino acids and positively charged residues at the C terminus. (B) Proposed model for the cell wall sorting reaction. The full-length surface protein precursor is secreted through the membrane via an N-terminal signal sequence. A charged tail at the C terminus of the protein may serve as a stop transfer signal. Following cleavage of this secretion signal, a sortase enzyme cleaves the protein between the threonine and glycine residues of the LPXTG motif, forming a thioacyl-enzyme intermediate. The free amine of the cell wall crossbridge of lipid II is deprotonated in the SrtA active site and serves as the acceptor for the transpeptidation reaction. The covalently linked proteinlipid II intermediate may then serve as a substrate for the transglycosylation reaction of cell wall synthesis.

(A) Diagram showing the structure of the second substrate of S. aureus SrtA, lipid II [undecaprenyl-pyrophosphate-MurNAc(-l-Ala–d-iGln–l-Lys(NH2-Gly5)–d-Ala-d-Ala)-β1→4-GlcNAc)]. The peptide of MurNAc is “branched” by forming a peptide bond between the ε-amine of lysine and a pentaglycine peptide. The terminal glycine α- amine serves as a nucleophile and forms a peptide bond to the carbonyl carbon of the threonine residue within the LPXTG motif. (B) Structure of the cell wall crossbridge. In a 4→3 linkage, a diamino acid (DA; either l-Lys or m-Dpm) at position 3 is linked through a peptide bond to the carboxyl of the d-Ala in the position 4 of a neighboring glycan chain, releasing the terminal d-Ala (position 5). These reactions are performed by cell wall transpeptidases (PBPs). The chemical nature of the diamino acid and crossbridge peptides can vary among different bacteria. (C) Cell wall crossbridges of different bacterial strains. In L. monocytogenes, bacilli, and corynebacteria, the cell wall peptides are unbranched and the diamino acid m-Dpm is directly linked to the d-Ala of the neighboring glycan chain.

Comparison of the primary and tertiary structures of SrtA and SrtB from S. aureus. (A) Ribbon diagrams of the folds of SrtA (PDB ID, 1IJA; residues 60 to 206) ( Ilangovan et al., 2001 ) and SrtB (PDB ID, 1NG5; residues 35 to 244) (Zhang et al., submitted). Both are presented in a similar orientation. The β-barrels of both structures align with a root mean square deviation of 3.2 Å. (B) Sequence alignment of SrtA and SrtB. Similar residues are shaded gray, and the catalytic cysteine and histidine residues are shaded black. Secondary-structure elements are indicated by arrows and cylinders for β-strands and α-helices, respectively. The N termini of both proteins (residues 1 to 59 in SrtA and 1 to 34 in SrtB) are absent in their respective structures and most probably contain a membrane anchor.

(A) Ribbon drawing of the structure of SrtAΔN59 (residues 60 to 206 of the S. aureus SrtA protein). Beginning at the N terminus, the β1 strand (G74 to I78) is followed by a short hairpin and lies antiparallel to the β2 strand (I83 to Y88). The β2 and β3 strands (V101 to A104) are positioned in parallel and are tethered by a 310 helix (P94 to L97), which crosses over the surface of the enzyme to form the lateral wall of the active site. The β3 and β4 strands (Q113 to G119) lie antiparallel with respect to one another and are followed by a long loop and a second α-helix, which assume a circuitous path to position the β5 strand (S140 to V146) for antiparallel alignment with the β1 strand. The β6 strand (E149 to K155) is then connected by a short hairpin turn for antiparallel pairing and followed by a long loop structure to connect it to the β7 strand (K177 to T183), which is aligned in parallel with β4. The active site sulfhydryl, C184, is positioned at the end of the β7 strand, which includes the LITC signature sequence of sortase enzymes. The structure is completed by a loop (D185 to W194), which connects the β7 and β8 strands (E195 to F200) for antiparallel pairing. Three acidic side chains (E105, E108, and D112) are poised to bind calcium, as judged by localized large-amplitude calcium-dependent changes in their chemical shifts and in surrounding amino acids. Additional residues in the β6-β7 loop displayed calcium-dependent chemical shift changes and may constitute a weaker Ca2+-binding site (spheres). The active-site side chains of H120, C184, and W194 are shown for reference. (B) Expanded view of the active site. H120 and C184 in sortase form a catalytic dyad that mediates the transpeptidation reaction and are positioned within a large hydrophobic pocket suitable for sorting-signal binding. A tryptophan (W194) is positioned in the active site and may play a limited role in catalysis ( Ton-That et al., 2002 ).

Proposed chemical mechanism of the SrtA sortase. Like the cysteine protease papain, catalysis presumably proceeds through the formation of a thioacyl intermediate. As the LPXTG substrate enters the active site-pocket, H120 functions as a general base, withdrawing a proton from C184 (step 1). The C184 nucleophile attacks the carbonyl carbon of threonine, and, proceeding through a tetrahedral intermediate (TH1) (step 2), results in a thioacyl-enzyme intermediate (step 3). The N-terminal amine of the pentaglycine crossbridge of lipid II then enters the active-site pocket (step 4), serving as an acceptor for the acyl-enzyme intermediate. Deacylation proceeds through a second tetrahedral intermediate (TH2) (step 5), resulting in the formation of a new peptide bond between the threonine of the LPXTG substrate and the terminal glycine of lipid II (step 6).

(A) The position-specific frequency of amino acids within the predicted sorting signals of sortases from different subfamilies. The one-letter symbol for the amino acid residue is given for each position in the six-residue motif. The size of each letter is proportional to the frequency with which an amino acid occurs in each position of the sorting-signal motif in the set of Cws-containing proteins that are predicted to be processed by a particular sortase type. If an amino acid appears in fewer than 8% of the substrates, the letter does not appear in the figure. A single amino acid at any position indicates >92% conservation. When no amino acid type is predominant in a given position of the motif, then the amino acid types found in the motif are given in brackets. (B) Pie chart showing the distribution of sortase homologs in gram-positive bacteria. A total of 176 sortase homologs were identified in gram-positive bacteria: 42 SrtA, 17 SrtB, 54 subfamily 3, 13 subfamily 4, and 14 subfamily 5 sortases. The remaining sortases could not be readily classified into a particular subfamily. (C) Pie chart showing the fraction of the 892 Cws-containing proteins that are predicted to be anchored by different types of sortases. A total of 203 Cws-containing proteins were not assigned to a specific sortase.

Molecular structures of sortase inhibitors. (A to C) structures that contain the LPXTG substrate mimic but replace the labile T-G peptide bond with a diazomethane (A), chloromethane (B), or vinyl sulfone (C) group for irreversible modification of the C184 nucleophile. (D) The phosphinate octapeptide NH2-ALPEAΨ(PO2HCH2)GEE-OH is a transition state mimic of the T-G scissile bond. (E) The natural SrtA inhibitor β-sitosterol-3-O-glucopyransoside from bulbs of F. verticillata.