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...

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The pathogen Staphylococcus aureus uses iron-regulated surface determinant (Isd) proteins to scavenge the essential nutrient iron from host hemoproteins. . High affinity binding to these structurally unrelated proteins requires residues located within a conserved aromatic motif that is positioned at the end of the ␤-barrel structure. Interestingly, this site is quite malleable, as other NEAT domains use it to bind heme. We also demonstrate that the IsdC NEAT domain can capture heme directly from Hb, suggesting that there are multiple pathways for heme transfer across the cell wall.

Staphylococcus aureus scavenges heme-iron from host hemoproteins using iron-regulated surface determinant (Isd) proteins. IsdC is the central conduit through which heme is passed across the cell wall and binds this molecule using a NEAr Transporter (NEAT) domain. NMR spectroscopy was used to determine the structure of IsdC in complex with a heme analog, zinc-substituted protoporphyrin IX (ZnPPIX). The backbone coordinates of the ensemble of conformers representing the structure exhibit a root mean square deviation to the mean structure of 0.53 ؎ 0.11 Å . IsdC partially buries protoporphyrin within a large hydrophobic pocket that is located at the end of its ␤-barrel structure. The central metal ion of the analog adopts a pentacoordinate geometry in which a highly conserved tyrosine residue serves as a proximal ligand. Consistent with the structure and its role in heme transfer across the cell wall, we show that IsdC weakly binds heme (K D ‫؍‬ 0.34 ؎ 0.12 M) and that ZnPPIX rapidly dissociates from the protein at a rate of 126 ؎ 30 s ؊1. NMR studies of the apo-form of IsdC reveal that a 3 10 helix within the binding pocket undergoes a flexible to rigid transition as heme is captured. This structural plasticity may increase the efficiency of heme transfer across the cell wall by facilitating protein-protein interactions between apoIsdC and upstream hemoproteins.Staphylococcus aureus is an opportunistic Gram-positive pathogen that causes lethal infections such as toxic shock syndrome, meningitis, and endocarditis (1, 2). The bacterium needs the essential nutrient iron to grow and although the human body contains large quantities of this metal, little is directly available to S. aureus as it is sequestered intracellularly (3) or bound to transferrin and lactoferrin (4, 5). During infections, S. aureus procures iron from heme (protoporphyrin IX ϩ iron), which contains ϳ80% of the total iron in the body (6). Heme-loaded hemoglobin (Hb) 5 is released into the blood plasma by the action of microbial hemolysins that rupture erythrocytes (7). A group of newly discovered proteins called iron-regulated surface determinant (Isd) proteins then scavenge heme and transfer it into the cytoplasm where it is degraded to liberate iron (8, 9). The heme-binding IsdC protein plays an essential role in the transfer of heme across the cell wall peptidylglycan (6, 10). Proteins homologous to IsdC are also present in a number of other important human pathogens (Bacillus anthracis and Listeria monocytogenes) (6, 11). Therefore, compounds that inhibit their ability to capture heme may be useful antibiotics.In Gram-negative bacteria, the process of heme-iron acquisition is reasonably well understood. Heme is captured from hemoproteins or hemophore-hemoprotein complexes by specific outer membrane receptors (5). It is then transferred into the periplasm in a Ton-B-dependent manner, where it is moved across the inner membrane by specific ABC-dependent permeases (5). Heme acquisition mechanisms used by Gram-positive bacteria are only beginning...

Hepatitis C virus (HCV) infection has been clinically associated with serum lipid abnormalities, yet our understanding of the effects of HCV on host lipid metabolism and conversely the function of individual lipids in HCV replication remains incomplete. Using liquid chromatography-mass spectrometry (LC-MS) metabolite profiling of the HCV JFH1 cell culture infection model, we identified a significant steady state accumulation of desmosterol, an immediate precursor to cholesterol. Pharmacological inhibition or RNAi-mediated depletion of DHCR7 significantly reduced steady-state HCV protein expression and viral genomic RNA. Moreover, this effect was reversed when cultures were supplemented with exogenous desmosterol. Together, these observations suggest an intimate connection between HCV replication and desmosterol homeostasis and that the enzymes responsible for synthesis of desmosterol may be novel targets for anti-viral design.