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...
The structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the trypanosomatid parasite Leishmania mexicana has been determined by X-ray crystallography. The protein crystallizes in space group P2(1)2(1)2(1) with unit cell parameters a = 99.0 A, b = 126.5 A, and c = 138.9 A. There is one 156,000 Da protein tetramer per asymmetric unit. The model of the protein with bound NAD+s and phosphates has been refined against 86% complete data from 10.0 to 2.8 A to a crystallographic Rfactor of 0.198. Density modification by noncrystallographic symmetry averaging was used during model building. The final model of the L. mexicana GAPDH tetramer shows small deviations of less than 0.5 degrees from ideal 222 molecular symmetry. The structure of L. mexicana GAPDH is very similar to that of glycosomal GAPDH from the related trypanosomatid Trypanosoma brucei. A significant structural difference between L. mexicana GAPDH and most previously determined GAPDH structures occurs in a loop region located at the active site. This unusual loop conformation in L. mexicana GAPDH occludes the inorganic phosphate binding site which has been seen in previous GAPDH structures. A new inorganic phosphate position is observed in the L. mexicana GAPDH structure. Model building studies indicate that this new anion binding site is well situated for nucleophilic attack of the inorganic phosphate on the thioester intermediate in the GAPDH-catalyzed reaction. Since crystals of L. mexicana GAPDH can be grown reproducibly and diffract much better than those of T. brucei GAPDH, L. mexicana GAPDH will be used as a basis for structure-based drug design targeted against trypanosomatid GAPDHs.
␣B-crystallin, a member of the small heat-shock protein family and a major eye lens protein, is a high molecular mass assembly and can act as a molecular chaperone. We report a synchrotron radiation x-ray solution scattering study of a truncation mutant from the human ␣B-crystallin (␣B57-157), a dimeric protein that comprises the ␣-crystallin domain of the ␣B-crystallin and retains a significant chaperone-like activity. According to the sequence analysis (more than 23% identity), the monomeric fold of the ␣-crystallin domain should be close to that of the small heat-shock protein from Methanococcus jannaschii (MjHSP16.5). The theoretical scattering pattern computed from the crystallographic model of the dimeric MjHSP16.5 deviates significantly from the experimental scattering by the ␣-crystallin domain, pointing to different quaternary structures of the two proteins. A rigid body modeling against the solution scattering data yields a model of the ␣-crystallin domain revealing a new dimerization interface. The latter consists of a strand-turn-strand motif contributed by each of the monomers, which form a four-stranded, antiparallel, intersubunit composite -sheet. This model agrees with the recent spin labeling results and suggests that the ␣B-crystallin is composed by flexible building units with an extended surface area. This flexibility may be important for biological activity and for the formation of ␣B-crystallin complexes of variable sizes and compositions.␣A-and ␣B-crystallin, which share 54% amino acid sequence identity, build the subunits of ␣-crystallin, a major eye lens protein, comprising up to 40% of the total lens proteins. The structural function of the ␣-crystallin is to assist in maintaining transparency in the lens (1). The chaperone-like function of ␣B-crystallin helps to avoid formation of large light-scattering aggregates and possibly helps to prevent cataract in the lens. Moreover, neurodegenerative diseases, ischemia, or multiple sclerosis lead to an overexpression of this protein, which makes it an object of special medical interest (2).The ␣-crystallin as well as other mammalian small heat-shock proteins (sHSPs)1 form large globular complexes with a diameter of about 10 -25 nm. Cryoelectron microscopy and image analysis revealed that ␣B-crystallin is a hollow spherical shell with variable quaternary structure (3), and a frequent exchange of subunits between the particles was observed. The chaperone activity of ␣B-crystallin is associated with partial perturbation of the substrate protein tertiary structure, leading to a multimeric molten globule-like state with increased hydrophobicity (4, 5). The exposed hydrophobic regions of ␣-crystallin interact with substrate proteins possessing an increased surface hydrophobicity but a low degree of unfolding (6).The molecular structure and subunit interactions in ␣-crystallin have long been under investigation. The stretches of residues promoting formation of lower or higher molecular weight ␣-crystallin oligomers have been identified. Upon additio...
The bound conformations of these receptor antagonist compounds preserve the toxin-galactose interactions previously observed for toxin-sugar complexes, but gain additional favorable interactions. The highest affinity compound, MNPG, is notable in that it displaces a water molecule that is observed to be well-ordered in all other previous and current crystal structures of toxin-sugar complexes. This could be a favorable entropic factor contributing to the increased affinity. The highest affinity members of the present set of antagonists (MNPG and TDG) bury roughly half (400 A2) of the binding-site surface covered by the full receptor GM1 pentasaccharide, despite being considerably smaller. This provides an encouraging basis for the creation of subsequent generations of derived compounds that can compete effectively with the natural receptor.
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