The Structure of the Staphylococcus aureus Sortase-Substrate Complex Reveals How the Universally Conserved LPXTG Sorting Signal Is Recognized

In Gram-positive bacteria, sortase enzymes assemble surface proteins and pili in the cell wall envelope. Sortases catalyze a transpeptidation reaction that joins a highly conserved LPXTG sorting signal within their polypeptide substrate to the cell wall or to other pilin subunits. The molecular basis of transpeptidation and sorting signal recognition are not well understood, because the intermediates of catalysis are short lived. We have overcome this problem by synthesizing an analog of the LPXTG signal whose stable covalent complex with the enzyme mimics a key thioacyl catalytic intermediate. Here we report the solution structure and dynamics of its covalent complex with the Staphylococcus aureus SrtA sortase. In marked contrast to a previously reported crystal structure, we show that SrtA adaptively recognizes the LPXTG sorting signal by closing and immobilizing an active site loop. We have also used chemical shift mapping experiments to localize the binding site for the triglycine portion of lipid II, the second substrate to which surface proteins are attached. We propose a unified model of the transpeptidation reaction that explains the functions of key active site residues. Since the sortase-catalyzed anchoring reaction is required for the virulence of a number of bacterial pathogens, the results presented here may facilitate the development of new anti-infective agents.Bacterial surface proteins function as virulence factors that enable pathogens to adhere to sites of infection, evade the immune response, acquire essential nutrients, and enter host cells (1). Gram-positive bacteria use a common mechanism to covalently attach proteins to the cell wall. This process is catalyzed by sortase transpeptidase enzymes, which join proteins bearing a highly conserved Leu-Pro-X-Thr-Gly (LPXTG, where X is any amino acid) sorting signal to the cross-bridge peptide of the peptidylglycan (2–4). Sortases also polymerize proteins containing sorting signals into pili, filamentous surface exposed structures that promote bacterial adhesion (5, 6). The search for small molecule sortase inhibitors is an active area of research, since these enzymes contribute to the virulence of a number of important pathogens, including among others Staphylococcus aureus, Listeria monocytogenes, Streptococcus pyogenes, and Streptococcus pneumoniae (reviewed in Refs. 7 and 8). Sortase enzymes are also promising molecular biology reagents that can be used to site-specifically attach proteins to a variety of biomolecules (9–14, 72).The sortase A (SrtA)⁷ enzyme from S. aureus is the prototypical member of the sortase enzyme family (15, 16). It anchors proteins to the murein sacculus that possess a COOH-terminal cell wall sorting signal that consists of a LPXTG motif, followed by a hydrophobic segment of amino acids and a tail composed of mostly positively charged residues (17). SrtA is located on the extracellular side of the membrane. After partial secretion of its protein substrate across the cell membrane, SrtA cleaves the LPXTG motif between the threonine and glycine residues, forming a thioacyl-linked protein-sortase intermediate (16). It then catalyzes the formation of an amide bond between the carboxyl group of the threonine and the cell wall precursor molecule lipid II (undecaprenyl-pyrophosphate-MurNAc(-l-Ala-d-iGln-l-Lys(NH₂-Gly₅)-d-Ala-d-Ala)-β1–4-GlcNAc)), creating a protein-lipid II-linked product that is incorporated into the peptidylglycan via the transglycosylation and transpeptidation reactions of bacterial cell wall synthesis (18–20). Over 900 sortase-attached proteins in 72 different strains of bacteria have thus far been identified (21, 22). The vast majority of these proteins contain a COOH-terminal sorting signal harboring an LPXTG motif and are anchored to the cell wall by enzymes closely related to SrtA.In vitro studies of SrtA have begun to define the mechanism of transpeptidation. SrtA consists of two parts: an unstructured amino-terminal tail that contains a stretch of nonpolar residues that embed it in the membrane and an autonomously folded catalytic domain that competently performs the transpeptidation reaction in vitro (SrtA_ΔN59, residues 60–206) (16, 23–25). Catalysis occurs through a ping-pong mechanism that is initiated when the thiol group of amino acid Cys¹⁸⁴ nucleophilically attacks the carbonyl carbon of the threonine residue within the LPXTG sorting signal (16, 23–25). This forms a transient tetrahedral intermediate that, upon breakage of the threonine-glycine peptide bond, rearranges into a more stable thioacyl enzyme-substrate linkage. SrtA then joins the terminal amine group within the pentaglycine branch of lipid II to the carbonyl carbon of the threonine, creating a second tetrahedral intermediate that is resolved into the lipid II-linked protein product (23).Sortase enzymes contain three conserved residues within their active sites: His¹²⁰, Cys¹⁸⁴, and Arg¹⁹⁷ (SrtA numbering). These residues play a critical role in catalysis, since their mutation in SrtA causes severe reductions in enzyme activity (16, 26–30). Although it is well established that Cys¹⁸⁴ forms a covalent linkage to the sorting signal, the functions of His¹²⁰ and Arg¹⁹⁷ are controversial. A variety of disparate functions have been ascribed to Arg¹⁹⁷. These include deprotonating Cys¹⁸⁴ (28), deprotonating lipid II (31), or stabilizing the binding of either the LPXTG sorting signal (28, 32) or oxyanion intermediates (31, 32). Different functions have also been proposed for His¹²⁰. Originally, it was suggested that it activated Cys¹⁸⁴ by forming an imidazolium-thiolate ion pair (26). However, subsequent pK_a measurements revealed that both His¹²⁰ and Cys¹⁸⁴ are predominantly uncharged at physiological pH values, leading to the suggestion that His¹²⁰ functions as a general base during catalysis (33). Most recently, it has been proposed that the most active form of the enzyme contains His¹²⁰ and Cys¹⁸⁴ in their charged states but that only a small fraction of SrtA exists in this form (～0.06%) prior to binding to the sorting signal (25).NMR and crystal structures of SrtA_ΔN59 have revealed that it adopts an eight-stranded β-barrel fold (31, 34). Other sortase enzymes have also been shown to possess a similar overall structure, including SrtB from S. aureus (27, 35), SrtB from Bacillus anthracis (27, 36), SrtA from S. pyogenes (37), and the SrtC-1 and SrtC-3 enzymes from S. pneumoniae (38). However, the molecular basis of substrate recognition remains poorly understood, because all of the structures reported to date have not contained a sorting signal bound to the enzyme. The lone exception is the crystal structure of SrtA_ΔN59 bound to an LPETG peptide (31). However, in this structure the peptide substrate is bound nonspecifically (see below) (32, 39).In this paper, we report the structure and dynamics of SrtA covalently bound to an analog of the LPXTG sorting signal. The structure of the complex resembles the thioacyl intermediate of catalysis, providing insights into the molecular basis of binding of the LPXTG sorting signal and the functions of key active site residues. Notably, the mechanism of substrate binding visualized in the NMR structure differs substantially from a previously reported crystal structure of SrtA_ΔN59 non-covalently bound to a LPETG peptide (31). We have also used NMR chemical shift mapping experiments to localize the binding site for a triglycine cell wall substrate analog. A mechanism of transpeptidation compatible with these new data is proposed.