SummaryIn Gram-positive bacteria proteins are displayed on the cell surface using sortase enzymes. These cysteine transpeptidases join proteins bearing an appropriate sorting signal to strategically positioned amino groups on the cell surface. Working alone, or in concert with other enzymes, sortases either attach proteins to the cross-bridge peptide of the cell wall or they link proteins together to form pili. Because surface proteins play a fundamental role in microbial physiology and are frequently virulence factors, sortase enzymes have been intensely studied since their discovery a little more than a decade ago. Based on their primary sequences and functions sortases can be partitioned into distinct families called class A to F enzymes. Most bacteria elaborate their surfaces using more than one type of sortase that function non-redundantly by recognizing unique sorting signals within their protein substrates. Here we review what is known about the functions of these enzymes and the molecular basis of catalysis. Particular emphasis is placed on 'pilin' specific class C sortases that construct structurally complex pili. Exciting new data have revealed that these enzymes are amazingly promiscuous in the substrates that they can employ and that there is a startling degree of diversity in their mechanism of action. We also review recent data that suggest that sortases are targeted to specific sites on the cell surface where they work with other sortases and accessory factors to properly function.

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 (His 126 ). 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 His 126 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.

Human immunodeficiency virus type 1 (HIV-1T o establish an infection after entry into a susceptible cell, human immunodeficiency virus type 1 (HIV-1) has to reverse transcribe its RNA genome to double-stranded DNA, followed by integration into the host genome. Reverse transcriptase (RT) and integrase (IN) are the viral enzymes responsible for catalyzing the essential steps of reverse transcription and integration, respectively. Both enzymes are synthesized as part of the Gag-Pol polyprotein, which is later processed by the viral protease to produce active RT and IN during HIV-1 maturation (1, 2). RT is a heterodimeric enzyme consisting of 66-and 51-kDa subunits and catalyzes the RNA-and DNA-dependent reverse transcription of the viral RNA genome into double-stranded cDNA through a complex cascade of events (3, 4). The 32-kDa IN has three domains: an N-terminal zinc-binding domain, a catalytic core domain, and a C-terminal domain (CTD) that binds DNA nonspecifically. IN catalyzes the integration of the viral cDNA into the host genome in two steps: an initial 3=-end processing step that removes two nucleotides at each 3= end and exposes a highly conserved CA 5= overhang, followed by a strand transfer step that inserts both processed viral DNA ends into the host cell genome (5, 6). In vitro, IN can also catalyze a reverse reaction, termed disintegration, resolving a DNA mimic of the viral-host DNA intermediate to products corresponding to a 3= processed viral DNA end and a target duplex DNA (7). IN can multimerize and forms a complex with viral DNA ends, termed the intasome (8-10). Structural studies of the prototype foamy virus (PFV) intasome found the tetramer to be the active IN configuration (9,11,12). HIV-1 IN has also been proposed to function as a tetramer (10,(13)(14)(15)(16)(17).Mutations in

Staphylococcus aureus is a leading cause of hospital-acquired infections in the United States and is a major health concern as methicillin-resistant S. aureus (MRSA) 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 analogs 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 analogs 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.

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