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.     

The Sortase A Enzyme That Attaches Proteins to the Cell Wall of Bacillus anthracis Contains an Unusual Active Site Architecture

The pathogen Bacillus anthracis uses the Sortase A (SrtA) enzyme to anchor proteins to its cell wall envelope during vegetative growth. To gain insight into the mechanism of protein attachment to the cell wall in B. anthracis we investigated the structure, backbone dynamics, and function of SrtA. The NMR structure of SrtA has been determined with a backbone coordinate precision of 0.40 ± 0.07 Å. SrtA possesses several novel features not previously observed in sortase enzymes including the presence of a structurally ordered amino terminus positioned within the active site and in contact with catalytically essential histidine residue (His¹²⁶). We propose that this appendage, in combination with a unique flexible active site loop, mediates the recognition of lipid II, the second substrate to which proteins are attached during the anchoring reaction. pK_a measurements indicate that His¹²⁶ is uncharged at physiological pH compatible with the enzyme operating through a “reverse protonation” mechanism. Interestingly, NMR relaxation measurements and the results of a model building study suggest that SrtA recognizes the LPXTG sorting signal through a lock-in-key mechanism in contrast to the prototypical SrtA enzyme from Staphylococcus aureus.     

Tempo and mode of early gene loss in endosymbiotic bacteria from insects

Background  

Understanding evolutionary processes that drive genome reduction requires determining the tempo (rate) and the mode (size and types of deletions) of gene losses. In this study, we analysed five endosymbiotic genome sequences of the gamma-proteobacteria (three different Buchnera aphidicola strains, Wigglesworthia glossinidia, Blochmannia floridanus) to test if gene loss could be driven by the selective importance of genes. We used a parsimony method to reconstruct a minimal ancestral genome of insect endosymbionts and quantified gene loss along the branches of the phylogenetic tree. To evaluate the selective or functional importance of genes, we used a parameter that measures the level of adaptive codon bias in E. coli (i.e. codon adaptive index, or CAI), and also estimates of evolutionary rates (Ka) between pairs of orthologs either in free-living bacteria or in pairs of symbionts.     

Cardiotoxicity of the cancer therapeutic agent imatinib mesylate

Imatinib mesylate (Gleevec) is a small-molecule inhibitor of the fusion protein Bcr-Abl, the causal agent in chronic myelogenous leukemia. Here we report ten individuals who developed severe congestive heart failure while on imatinib and we show that imatinib-treated mice develop left ventricular contractile dysfunction. Transmission electron micrographs from humans and mice treated with imatinib show mitochondrial abnormalities and accumulation of membrane whorls in both vacuoles and the sarco- (endo-) plasmic reticulum, findings suggestive of a toxic myopathy. With imatinib treatment, cardiomyocytes in culture show activation of the endoplasmic reticulum (ER) stress response, collapse of the mitochondrial membrane potential, release of cytochrome c into the cytosol, reduction in cellular ATP content and cell death. Retroviral gene transfer of an imatinib-resistant mutant of c-Abl, alleviation of ER stress or inhibition of Jun amino-terminal kinases, which are activated as a consequence of ER stress, largely rescues cardiomyocytes from imatinib-induced death. Thus, cardiotoxicity is an unanticipated side effect of inhibition of c-Abl by imatinib.     

NMR structure of the Tn916 integrase-DNA complex

The integrase protein catalyzes the excision and integration of the Tn916 conjugative transposon, a promiscuous genetic element that spreads antibiotic resistance in pathogenic bacteria. The solution structure of the N-terminal domain of the Tn916 integrase protein bound to its DNA-binding site within the transposon arm has been determined. The structure reveals an interesting mode of DNA recognition, in which the face of a three-stranded antiparallel beta-sheet is positioned within the major groove. A comparison to the structure of the homing endonuclease I-Ppol-DNA complex suggests that the three-stranded sheet may represent a new DNA-binding motif whose residue composition and position within the major groove are varied to alter specificity. The structure also provides insights into the mechanism of conjugative transposition. The DNA in the complex is bent approximately 35 degrees and may, together with potential interactions between bound integrase proteins at directly repeated sites, significantly bend the arms of the transposon.     

Sortase from Staphylococcus aureus does not contain a thiolate-imidazolium ion pair in its active site

Many surface proteins are anchored to the cell wall by the action of sortase enzymes, a recently discovered family of cysteine transpeptidases. As the surface proteins of human pathogens are frequently required for virulence, the sortase-mediated anchoring reaction represents a potential target for new anti-infective agents. It has been suggested that the sortase from Staphylococcus aureus (SrtA), may use a similar catalytic strategy as the papain cysteine proteases, holding its Cys184 side chain in an active configuration through a thiolate-imidazolium ion interaction with residue His120. To investigate the mechanism of transpeptidation, we have synthesized a peptidyl-vinyl sulfone substrate mimic that irreversibly inhibits SrtA. Through the study of the pH dependence of SrtA inhibition and NMR, we have estimated the pKas of the active site thiol (Cys184) and imidazole (His120) to be approximately 9.4 and 7.0, respectively. These measurements are inconsistent with the existence of a thiolate-imidazolium ion pair and suggest a general base catalysis mechanism during transpeptidation.     

The basal and major pilins in the Corynebacterium diphtheriae SpaA pilus adopt similar structures that competitively react with the pilin polymerase

Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the ^CdSrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that ^CdSrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (^NSpaA) that is also crosslinked by ^CdSrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed “latch” mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting ^NSpaA for access to a shared thioester enzyme–substrate reaction intermediate.     

Structural requirements of carbohydrates to bind Agaricus bisporus lectin

Galbeta1-3GalNAc (T-disaccharide) and related molecules were assayed to describe the structural requirements of carbohydrates to bind Agaricus bisporus lectin (ABL). Results provide insight into the most relevant regions of T-disaccharide involved in the binding of ABL. It was found that monosaccharides bind ABL weakly indicating a more extended carbohydrate-binding site as compared to those involvedin the T- disaccharide specific lectins such as jacalin and peanut agglutinin. Lacto-N-biose (Galbeta1-3GlcNAc) unlike T-disaccharide, is unable to inhibit the ABL interaction, thus showing the great importance of the position of the axial C-4 hydroxyl group of GalNAc in T-disaccharide. This finding could explain the inhibitory ability of Galbeta1-6GlcNAc and lactose because C-4 and C-3 hydroxyl groups of reducing Glc, respectively, occupy a similar position as reported by conformational analysis. From the comparison of different glycolipids bearing terminal T-disaccharide bound to different linkages, it can be seen than ABL binding is even more impaired by an adjacent C-6 residual position than by the anomeric influence of T-disaccharide. Furthermore, the addition of beta-GlcNAc to the terminal T-disaccharide in C-3 position of Gal does not affect the ABL binding whereas if an anionic group such as glucuronic acid is added to C-3, the binding is partially affected. These findings demonstrate that ABL holds a particular binding nature different from that of other T-disaccharide specific lectins.      

Sarcocystis falcatula of opossums: transmission by cockroaches with fatal pulmonary disease in psittacine birds.

Old World psittacines experienced an acute fatal illness in outdoor breeding collections in South Florida. Toxoplasma-like organisms were found histologically in pulmonary capillaries and elsewhere. Because the organisms underwent schizogony and could not be transmitted to mice, we looked for a cause other than Toxoplasma gondii. An opossum was trapped on the premises of 1 facility and was found to be shedding sporocysts similar to Sarcocystis falcatula in its feces. Cockroaches were prevalent and suspected as transport hosts. Cockroaches that had ingested opossum feces and subsequently were fed to cockatoos induced an identical fatal illness. Obstruction of pulmonary capillaries by developing schizonts and pulmonary edema were the most important pathologic findings. The epidemic was stopped by biological insect control employing flightless chickens to reduce cockroach populations and by an electric fence restricting access of opossums to these outdoor psittacine breeding facilities.