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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