Artem G. Evdokimov

Mechanism of insulin sensitization by BMOV (bis maltolato oxo vanadium); unliganded vanadium (VO4) as the active component

Organovanadium compounds have been shown to be insulin sensitizers in vitro and in vivo. One potential biochemical mechanism for insulin sensitization by these compounds is that they inhibit protein tyrosine phosphatases (PTPs) that negatively regulate insulin receptor activation and signaling. In this study, bismaltolato oxovanadium (BMOV), a potent insulin sensitizer, was shown to be a reversible, competitive phosphatase inhibitor that inhibited phosphatase activity in cultured cells and enhanced insulin receptor activation in vivo. NMR and X-ray crystallographic studies of the interaction of BMOV with two different phosphatases, HCPTPA (human low molecular weight cytoplasmic protein tyrosine phosphatase) and PTP1B (protein tyrosine phosphatase 1B), demonstrated uncomplexed vanadium (VO(4)) in the active site. Taken together, these findings support phosphatase inhibition as a mechanism for insulin sensitization by BMOV and other organovanadium compounds and strongly suggest that uncomplexed vanadium is the active component of these compounds.

Similar modes of polypeptide recognition by export chaperones in flagellar biosynthesis and type III secretion

Assembly of the bacterial flagellum and type III secretion in pathogenic bacteria require cytosolic export chaperones that interact with mobile components to facilitate their secretion. Although their amino acid sequences are not conserved, the structures of several type III secretion chaperones revealed striking similarities between their folds and modes of substrate recognition. Here, we report the first crystallographic structure of a flagellar export chaperone, Aquifex aeolicus FliS. FliS adopts a novel fold that is clearly distinct from those of the type III secretion chaperones, indicating that they do not share a common evolutionary origin. However, the structure of FliS in complex with a fragment of FliC (flagellin) reveals that, like the type III secretion chaperones, flagellar export chaperones bind their target proteins in extended conformation and suggests that this mode of recognition may be widely used in bacteria.

Differential effects of short affinity tags on the crystallization of Pyrococcus furiosus maltodextrin-binding protein

Pyrococcus furiosus maltodextrin-binding protein readily forms large orthorhombic crystals that diffract to high resolution. This protein was used as a model system to investigate the influence of five short affinity tags (His(6), Arg(5), Strep tag II, FLAG tag and the biotin acceptor peptide) on the formation of protein crystals and their ability to diffract X-rays. The results indicate that the amino-acid sequence of the tag can have a profound effect on both of these parameters. Consequently, the ability to obtain diffracting crystals of a particular protein may depend as much on which affinity tag is selected as it does on whether an affinity tag is used at all.

Structural basis for the fast maturation of Arthropoda green fluorescent protein

Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP‐like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non‐aggregating mutant of Copepoda fluorescent protein and solved its high‐resolution crystal structure. It was found that the protein β‐barrel contains a pore, leading to the chromophore. Using site‐directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore.

Structural basis for effectiveness of siderophore-conjugated monocarbams against clinically relevant strains of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes nosocomial infections for which there are limited treatment options. Penicillin-binding protein PBP3, a key therapeutic target, is an essential enzyme responsible for the final steps of peptidoglycan synthesis and is covalently inactivated by β-lactam antibiotics. Here we disclose the first high resolution cocrystal structures of the P. aeruginosa PBP3 with both novel and marketed β-lactams. These structures reveal a conformational rearrangement of Tyr532 and Phe533 and a ligand-induced conformational change of Tyr409 and Arg489. The well-known affinity of the monobactam aztreonam for P. aeruginosa PBP3 is due to a distinct hydrophobic aromatic wall composed of Tyr503, Tyr532, and Phe533 interacting with the gem-dimethyl group. The structure of MC-1, a new siderophore-conjugated monocarbam complexed with PBP3 provides molecular insights for lead optimization. Importantly, we have identified a novel conformation that is distinct to the high-molecular-weight class B PBP subfamily, which is identifiable by common features such as a hydrophobic aromatic wall formed by Tyr503, Tyr532, and Phe533 and the structural flexibility of Tyr409 flanked by two glycine residues. This is also the first example of a siderophore-conjugated triazolone-linked monocarbam complexed with any PBP. Energetic analysis of tightly and loosely held computed hydration sites indicates protein desolvation effects contribute significantly to PBP3 binding, and analysis of hydration site energies allows rank ordering of the second-order acylation rate constants. Taken together, these structural, biochemical, and computational studies provide a molecular basis for recognition of P. aeruginosa PBP3 and open avenues for future design of inhibitors of this class of PBPs.

Crystal structure of the Yersinia pestis GTPase activator YopE

Yersinia pestis, the causative agent of bubonic plague, evades the immune response of the infected organism by using a type III (contact‐dependent) secretion system to deliver effector proteins into the cytosol of mammalian cells, where they interfere with signaling pathways that regulate inflammation and cytoskeleton dynamics. The cytotoxic effector YopE functions as a potent GTPase‐activating protein (GAP) for Rho family GTP‐binding proteins, including RhoA, Rac1, and Cdc42. Down‐regulation of these molecular switches results in the loss of cell motility and inhibition of phagocytosis, enabling Y. pestis to thrive on the surface of macrophages. We have determined the crystal structure of the GAP domain of YopE (YopEGAP; residues 90–219) at 2.2‐Å resolution. Apart from the fact that it is composed almost entirely of α‐helices, YopEGAP shows no obvious structural similarity with eukaryotic RhoGAP domains. Moreover, unlike the catalytically equivalent arginine fingers of the eukaryotic GAPs, which are invariably contained within flexible loops, the critical arginine in YopEGAP (Arg144) is part of an α‐helix. The structure of YopEGAP is strikingly similar to the GAP domains from Pseudomonas aeruginosa (ExoSGAP) and Salmonella enterica (SptPGAP), despite the fact that the three amino acid sequences are not highly conserved. A comparison of the YopEGAP structure with those of the Rac1‐ExoSGAP and Rac1‐SptP complexes indicates that few, if any, significant conformational changes occur in YopEGAP when it interacts with its G protein targets. The structure of YopEGAP may provide an avenue for the development of novel therapeutic agents to combat plague.

Three-dimensional structure of the type III secretion chaperone SycE from Yersinia pestis

Many bacterial pathogens utilize a type III (contact-dependent) secretion system to inject cytotoxic effector proteins directly into host cells. This ingenious mechanism, designed for both bacterial offense and defense, has been studied most extensively in Yersinia spp. To be exported efficiently, at least three of the effectors (YopE, YopH and YopT) and several other proteins that transit the type III secretion pathway in Yersinia (YopN, YopD and YopB) must first form transient complexes with cognate-specific Yop chaperone (Syc) proteins. The cytotoxic effector YopE, a selective activator of mammalian Rho-family GTPases, associates with SycE. Here, the structure of Y. pestis SycE at 1.95A resolution is reported. SycE possesses a novel fold with an unusual dimerization motif and an intriguing basic cavity located on the dyad axis of the dimer that may participate in its interaction with YopE.

MALDI-TOF MS as a label-free approach to rapid inhibitor screening

Mass Spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays. These measurements are typically performed by time-consuming LC-MS to eliminate buffer salts that interfere with electrospray ionization MS. However, matrix-assisted laser desorption ionization, time-of-flight MS (MALDI-TOF MS) offers a label-free and direct readout of substrate and product, a fast sampling rate, and is tolerant of many buffer salts, reagents, and compounds that are typically found in enzyme reaction mixtures. In this report, a demonstration of how MALDI-TOF MS can be used to directly measure ratios of substrates and products to produce IC50 curves for rapid enzyme assays and compound screening is provided. Typical reproducibility parameters were <7% RSD—a value comparable to ESI MS quantitative assays and well within the acceptable limits for screening assays. The speed of the MALDI readout is currently about 10 s per sample, thus allowing for over 7500 samples/day. From a simplicity standpoint, the enzymatic reaction mixtures are prepared by liquid handling robots, the reactions are stopped by addition of a 10 times volume of acidic matrix solution, and the samples are simultaneously transferred to MALDI target plate for analysis. Importantly, the ratios of substrate to product are of sufficient reproducibility to eliminate the need for internal standards and, thus, minimize the cost and increasing the speed of assay development.

Discovery of Brain-Penetrant, Irreversible Kynurenine Aminotransferase II Inhibitors for Schizophrenia.

Kynurenine aminotransferase (KAT) II has been identified as a potential new target for the treatment of cognitive impairment associated with schizophrenia and other psychiatric disorders. Following a high-throughput screen, cyclic hydroxamic acid PF-04859989 was identified as a potent and selective inhibitor of human and rat KAT II. An X-ray crystal structure and (13)C NMR studies of PF-04859989 bound to KAT II have demonstrated that this compound forms a covalent adduct with the enzyme cofactor, pyridoxal phosphate (PLP), in the active site. In vivo pharmacokinetic and efficacy studies in rat show that PF-04859989 is a brain-penetrant, irreversible inhibitor and is capable of reducing brain kynurenic acid by 50% at a dose of 10 mg/kg (sc). Preliminary structure-activity relationship investigations have been completed and have identified the positions on this scaffold best suited to modification for further optimization of this novel series of KAT II inhibitors.

Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase

Bacterial acetyl‐CoA carboxylase is a multifunctional biotin‐dependent enzyme that consists of three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Acetyl‐CoA carboxylase is a potentially attractive target for novel antibiotics because it catalyzes the first committed step in fatty acid biosynthesis. In the first half‐reaction, BC catalyzes the ATP‐dependent carboxylation of BCCP. In the second half‐reaction, the carboxyl group is transferred from carboxybiotinylated BCCP to acetyl‐CoA to produce malonyl‐CoA. A series of structures of BC from several bacteria crystallized in the presence of various ATP analogs is described that addresses three major questions concerning the catalytic mechanism. The structure of BC bound to AMPPNP and the two catalytically essential magnesium ions resolves inconsistencies between the kinetics of active‐site BC mutants and previously reported BC structures. Another structure of AMPPNP bound to BC shows the polyphosphate chain folded back on itself, and not in the correct (i.e., extended) conformation for catalysis. This provides the first structural evidence for the hypothesis of substrate‐induced synergism, which posits that ATP binds nonproductively to BC in the absence of biotin. The BC homodimer has been proposed to exhibit half‐sites reactivity where the active sites alternate or “flip‐flop” their catalytic cycles. A crystal structure of BC showed the ATP analog AMPPCF2P bound to one subunit while the other subunit was unliganded. The liganded subunit was in the closed or catalytic conformation while the unliganded subunit was in the open conformation. This provides the first structural evidence for half‐sites reactivity in BC.