Brendan R. Amer

Discovery of Staphylococcus aureus sortase A inhibitors using virtual screening and the relaxed complex scheme.

Staphylococcus aureus is the leading cause of hospital‐acquired infections in the United States. The emergence of multidrug‐resistant strains of S. aureus has created an urgent need for new antibiotics. Staphylococcus aureus uses the sortase A enzyme to display surface virulence factors suggesting that compounds that inhibit its activity will function as potent anti‐infective agents. Here, we report the identification of several inhibitors of sortase A using virtual screening methods that employ the relaxed complex scheme, an advanced computer‐docking methodology that accounts for protein receptor flexibility. Experimental testing validates that several compounds identified in the screen inhibit the activity of sortase A. A lead compound based on the 2‐phenyl‐2,3‐dihydro‐1H‐perimidine scaffold is particularly promising, and its binding mechanism was further investigated using molecular dynamics simulations and conducting preliminary structure–activity relationship studies.

Structural and Computational Studies of the Staphylococcus aureus Sortase B-Substrate Complex Reveal a Substrate-stabilized Oxyanion Hole.

Background: Sortase enzymes catalyze a transpeptidation reaction that displays bacterial surface proteins. Results: Structural and computational studies reveal how the sortase B enzyme recognizes its sorting signal substrate. Conclusion: Sortase enzymes catalyze transpeptidation using a substrate-stabilized oxyanion hole. Significance: The results of this work could facilitate the rational design of sortase inhibitors. Sortase cysteine transpeptidases covalently attach proteins to the bacterial cell wall or assemble fiber-like pili that promote bacterial adhesion. Members of this enzyme superfamily are widely distributed in Gram-positive bacteria that frequently utilize multiple sortases to elaborate their peptidoglycan. Sortases catalyze transpeptidation using a conserved active site His-Cys-Arg triad that joins a sorting signal located at the C terminus of their protein substrate to an amino nucleophile located on the cell surface. However, despite extensive study, the catalytic mechanism and molecular basis of substrate recognition remains poorly understood. Here we report the crystal structure of the Staphylococcus aureus sortase B enzyme in a covalent complex with an analog of its NPQTN sorting signal substrate, revealing the structural basis through which it displays the IsdC protein involved in heme-iron scavenging from human hemoglobin. The results of computational modeling, molecular dynamics simulations, and targeted amino acid mutagenesis indicate that the backbone amide of Glu224 and the side chain of Arg233 form an oxyanion hole in sortase B that stabilizes high energy tetrahedral catalytic intermediates. Surprisingly, a highly conserved threonine residue within the bound sorting signal substrate facilitates construction of the oxyanion hole by stabilizing the position of the active site arginine residue via hydrogen bonding. Molecular dynamics simulations and primary sequence conservation suggest that the sorting signal-stabilized oxyanion hole is a universal feature of enzymes within the sortase superfamily.

NMR structure-based optimization of Staphylococcus aureus sortase A pyridazinone inhibitors

Staphylococcus aureus is a leading cause of hospital‐acquired infections in the USA and is a major health concern as methicillin‐resistant S. aureus and other antibiotic‐resistant strains are common. Compounds that inhibit the S. aureus sortase (SrtA) cysteine transpeptidase may function as potent anti‐infective agents as this enzyme attaches virulence factors to the bacterial cell wall. While a variety of SrtA inhibitors have been discovered, the vast majority of these small molecules have not been optimized using structure‐based approaches. Here we have used NMR spectroscopy to determine the molecular basis through which pyridazinone‐based small molecules inhibit SrtA. These inhibitors covalently modify the active cysteine thiol and partially mimic the natural substrate of SrtA by inducing the closure of an active site loop. Computational and synthetic chemistry methods led to second‐generation analogues that are ~70‐fold more potent than the lead molecule. These optimized molecules exhibit broad‐spectrum activity against other types of class A sortases, have reduced cytotoxicity, and impair SrtA‐mediated protein display on S. aureus cell surface. Our work shows that pyridazinone analogues are attractive candidates for further development into anti‐infective agents, and highlights the utility of employing NMR spectroscopy and solubility‐optimized small molecules in structure‐based drug discovery.

Solution structure of the PhoP DNA-binding domain from Mycobacterium tuberculosis

Tuberculosis caused by Mycobacterium tuberculosis is a leading cause of death world-wide. The PhoP protein is required for virulence and is part of the PhoPR two-component system that regulates gene expression. The NMR-derived solution structure of the PhoP C-terminal DNA-binding domain is reported. Residues 150 to 246 form a structured domain that contains a winged helix-turn-helix motif. We provide evidence that the transactivation loop postulated to contact RNA polymerase is partially disordered in solution, and that the polypeptide that connects the DNA-binding domain to the regulatory domain is unstructured.

A sweet new role for LCP enzymes in protein glycosylation.

The peptidoglycan that surrounds Gram‐positive bacteria is affixed with a range of macromolecules that enable the microbe to effectively interact with its environment. Distinct enzymes decorate the cell wall with proteins and glycopolymers. Sortase enzymes covalently attach proteins to the peptidoglycan, while LytR‐CpsA‐Psr (LCP) proteins are thought to attach teichoic acid polymers and capsular polysaccharides. Ton‐That and colleagues have discovered a new glycosylation pathway in the oral bacterium Actinomyces oris in which sortase and LCP enzymes operate on the same protein substrate. The A. oris LCP protein has a novel function, acting on the cell surface to transfer glycan macromolecules to a protein, which is then attached to the cell wall by a sortase. The reactions are tightly coupled, as elimination of the sortase causes the lethal accumulation of glycosylated protein in the membrane. Since sortase enzymes are attractive drug targets, this novel finding may provide a convenient cell‐based tool to discover inhibitors of this important enzyme family.

In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking

Significance Gram-positive sortase enzymes represent two broad functional categories—those that cross-link proteins to the cell wall and those that can catalyze this reaction and polymerize proteins to build adhesive pilus fibers. Here we report an in vitro reproduction of a robust pilus polymerization reaction using a variant of a corynebacterial pilus-specific sortase in which the catalytic center is unmasked. By molecular modeling, we uncovered a conserved structural element of pilus-specific sortases critical for protein ligation in vitro and further demonstrated that the activated sortase ligates the isolated domains of the pilin harboring the donor and acceptor motifs for ligation. Besides enabling future molecular studies and antibiotic development, our system provides a powerful platform for bioconjugation and protein engineering. Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction, first cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate and then joining the terminal Thr to the nucleophilic Lys residue residing within the pilin motif of another pilin protomer. To date, the determinants of class C enzymes that uniquely enable them to construct pili remain unknown. Here, informed by high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and utilizing a structural variant of the enzyme (SrtA2M), whose catalytic pocket has been unmasked by activating mutations, we successfully reconstituted in vitro polymerization of the cognate major pilin (SpaA). Mass spectrometry, electron microscopy, and biochemical experiments authenticated that SrtA2M synthesizes pilus fibers with correct Lys–Thr isopeptide bonds linking individual pilins via a thioacyl intermediate. Structural modeling of the SpaA–SrtA–SpaA polymerization intermediate depicts SrtA2M sandwiched between the N- and C-terminal domains of SpaA harboring the reactive pilin and LPXTG motifs, respectively. Remarkably, the model uncovered a conserved TP(Y/L)XIN(S/T)H signature sequence following the catalytic Cys, in which the alanine substitutions abrogated cross-linking activity but not cleavage of LPXTG. These insights and our evidence that SrtA2M can terminate pilus polymerization by joining the terminal pilin SpaB to SpaA and catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile platform for protein engineering and bio-conjugation that has major implications for biotechnology.

Protein Labeling via a Specific Lysine-Isopeptide Bond Using the Pilin Polymerizing Sortase from Corynebacterium diphtheriae

Proteins that are site-specifically modified with peptides and chemicals can be used as novel therapeutics, imaging tools, diagnostic reagents and materials. However, there are few enzyme-catalyzed methods currently available to selectively conjugate peptides to internal sites within proteins. Here we show that a pilus-specific sortase enzyme from Corynebacterium diphtheriae (CdSrtA) can be used to attach a peptide to a protein via a specific lysine-isopeptide bond. Using rational mutagenesis we created CdSrtA3M, a highly activated cysteine transpeptidase that catalyzes in vitro isopeptide bond formation. CdSrtA3M mediates bioconjugation to a specific lysine residue within a fused domain derived from the corynebacterial SpaA protein. Peptide modification yields greater than >95% can be achieved. We demonstrate that CdSrtA3M can be used in concert with the Staphylococcus aureus SrtA enzyme, enabling dual, orthogonal protein labeling via lysine-isopeptide and backbone-peptide bonds.