The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB.

Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine 3 phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine.Syk family members include two human proteins, Syk (spleen tyrosine kinase) and its closest relative Zap-70 (70-kDa chain-associated protein 1). Syk operates downstream of the B-cell receptor in B-cells, the IgE receptor Fc⑀RI in mast cells, Fc␥R in macrophages (2), and other receptors (3). Zap-70 performs a similar function in T-cell receptor signaling (4). Syk is expressed in a wide range of cell types, although its function is best understood in hematopoietic cells. Knock-out mouse studies have shown that Syk is essential for lymphocyte development (2). Syk has attracted particular interest as a therapeutic target for treatment of asthma, because Syk-deficient mast cells do not degranulate in response to Fc⑀RI aggregation (5, 6). Analyses of three-dimensional structures of the kinase domains of the insulin receptor kinase (7,8), fibroblast growth factor receptor 1 (9), and Lck (10) gave rise to a model of conformational transition upon activation in kinases. In this model, the preactivated conformation is characterized by activation loop occlusion of the ATP-and/or substrate-binding sites ("loop in"), effectively preventing substrate access. Phosphorylation stabilizes an open conformation of the activation loop ("loop out") that does not occlude the substrate-binding sites and is compatible with catalysis (11, 12). In the loop-out conformation, the activation loop also forms a platform for peptide substrate docking (12). In addition to permitting access to the su...

These structures provide support for a hypothesis that explains the in vivo action of LuxS. Specifically, acting as a homodimer, the protein binds a methionine analog, S-ribosylhomocysteine (SRH). The zinc atom is in position to cleave the ribose ring in a step along the synthesis pathway of AI-2.

The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation of MET by mutation or gene amplification has been linked to kidney, gastric, and lung cancers. In other cancers, such as glioblastoma, autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its exquisite selectivity for MET. SGX523 potently inhibited MET with an IC 50 of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation, and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition. [Mol Cancer Ther 2009;8(12):3181-90]

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We have begun a small scale structural genomics project aimed at obtaining fold information for the set of sequence families comprising eukaryotic intracellular signaling domains, and exploiting that information to understand biological function and mechanism. The SMART database (http://smart.embl-heidelberg.de/smart) is the primary target list for the project. The practical issues of obtaining soluble and crystallizable representatives for each sequence family will be discussed. The question of how much can be learned about signaling mechanisms from elucidating the structure of one representative protein per sequence family will be considered. Examples illustrating issues of principle and practice will be drawn from recent work on VHS and other domains. Acta Cryst. (2002 A Structural Genomics research program at CARB that seeks to infer the function of so-called hypothetical proteins from the structural information has yielded so far some 25 structures. The structures exhibit both previously known and novel folds. I will review the results provide specific examples and assess the power and limits of this approach. The genomes of Staphylococcus aureus and Streptococcus pyogenes, two important human pathogens, encode a variety of virulence factors, some of which have been clearly linked to disease. These include toxins such as staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin (TSST). These form a family of superantigens (SAgs) with a common fold and the ability to severely disrupt the human immune system by binding to both T-cell receptors and MHC class II molecules. Keywords: STRUCTURAL GENOMICS, SIGNAL TRANSDUCTION, PROTEIN TRANSPORT Keywords: STRUCTURAL GENOMICS HYPOTHETICAL PROTEINSThe Staphylococcus aureus genome also encodes a cluster of SAg-like genes whose functions are unknown. These genes are clustered on a putative pathogenicity island, implying a role in virulence. We crystallized one of these proteins, SET3, and solved and refined its structure at 1.9 Å resolution (R = 20.5%, Rfree = 24.0%). SET3 has the characteristic SAg-family fold, consistent with its sequence similarity (26% identity to TSST). Residues implicated in Tcell receptor and MHC binding in the SAgs are changed in SET3, however, and we have shown that SET3 does not have SAg activity. On the other hand, there are strong indications from seroconversion rates and antibody titres that SET3 has a role in pathogenicity. In the crystal, SET3 forms a dimer with a highly positively charged surface. Dimer formation occurs primarily through an extended β -hairpin that differentiates SET3 from other SAg-family proteins. We have shown that SET3 binds to DNA, which stabilizes SET3 dimer formation, but we hypothesize that the 'true' ligands for SET3 are likely to be negatively charged cell surface molecules. The high-throughput structure determination platform developed at Structural GenomiX is being utilized to increase the efficiency and effectiveness of the drug-discovery process. Parallelization increases the crystallization succes...

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