Crystal structures of TM0549 and NE1324--two orthologs of E. coli AHAS isozyme III small regulatory subunit

Crystal structures of two orthologs of the regulatory subunit of acetohydroxyacid synthase III (AHAS, EC 2.2.1.6) from Thermotoga maritima (TM0549) and Nitrosomonas europea (NE1324) were determined by single-wavelength anomalous diffraction methods with the use of selenomethionine derivatives at 2.3 A and 2.5 A, respectively. TM0549 and NE1324 share the same fold, and in both proteins the polypeptide chain contains two separate domains of a similar size. Each protein contains a C-terminal domain with ferredoxin-type fold and an N-terminal ACT domain, of which the latter is characteristic for several proteins involved in amino acid metabolism. The ferredoxin domain is stabilized by a calcium ion in the crystal structure of NE1324 and by a Mg(H2O)(6)2+ ion in TM0549. Both TM0549 and NE1324 form dimeric assemblies in the crystal lattice.     

Structure and ligand binding of the soluble domain of a Thermotoga maritima membrane protein of unknown function TM1634

As a part of the Joint Center for Structural Genomics (JCSG) biological targets, the structures of soluble domains of membrane proteins from Thermotoga maritima were pursued. Here, we report the crystal structure of the soluble domain of TM1634, a putative membrane protein of 128 residues (15.1 kDa) and unknown function. The soluble domain of TM1634 is an alpha-helical dimer that contains a single tetratrico peptide repeat (TPR) motif in each monomer where each motif is similar to that found in Tom20. The overall fold, however, is unique and a DALI search does not identify similar folds beyond the 38-residue TPR motif. Two different putative ligand binding sites, in which PEG200 and Co(2+) were located, were identified using crystallography and NMR, respectively.     

Crystallization, crystal structure analysis and preliminary molecular model of the bilin binding protein from the insect Pieris brassicae

The bilin binding protein of the butterfly Pieris brassicae has been prepared, crystallized and its crystal structure determined at high resolution using film and FAST area detector intensity data. The crystallographic asymmetric unit contains a tetramer of identical subunits with a molecular weight of about 90,000. The crystal structure was determined by isomorphous replacement. Use was made of the molecular symmetry to improve phases. A molecular interpretation of the electron density distribution and partial tracing of the polypeptide chain was possible without amino acid sequence information, as the fold is very similar to retinol binding protein. It is characterized by a beta-barrel formed by two orthogonal beta-sheets and an alpha-helix. The bilin pigment seems to be bound within the beta-barrel analogously to retinol in retinol binding protein. The tetramer in the crystal has C2 symmetry and is a dimer of dimers of quasi-equivalent subunits.     

Crystal structure of a hypothetical protein,TM841 of Thermotoga maritima,reveals its function as a fatty acid-binding protein

We determined the three-dimensional (3D) crystal structure of protein TM841, a protein product from a hypothetical open-reading frame in the genome of the hyperthermophile bacterium Thermotoga maritima, to 2.0 A resolution. The protein belongs to a large protein family, DegV or COG1307 of unknown function. The 35 kDa protein consists of two separate domains, with low-level structural resemblance to domains from other proteins with known 3D structures. These structural homologies, however, provided no clues for the function of TM841. But the electron density maps revealed clear density for a bound fatty-acid molecule in a pocket between the two protein domains. The structure indicates that TM841 has the molecular function of fatty-acid binding and may play a role in the cellular functions of fatty acid transport or metabolism.     

The Crystal Structure of Glutamine-binding Protein fromEscherichia coli

The crystal structure of the glutamine-binding protein (GlnBP) fromEscherichia coliin a ligand-free “open” conformational state has been determined by isomorphous replacement methods and refined to anR-value of 21.4% at 2.3 Å resolution. There are two molecules in the asymmetric unit, related by pseudo 4-fold screw symmetry. The refined model consists of 3587 non-hydrogen atoms from 440 residues (two monomers), and 159 water molecules. The structure has root-mean-square deviations of 0.013 Å from “deal” bond lengths and 1.5° from “ideal” bond angles.The GlnBP molecule has overall dimensions of approximately 60 Å × 40 Å × 35 Å and is made up of two domains (termed large and small), which exhibit a similar supersecondary structure, linked by two antiparallel β-strands. The small domain contains three α-helices and four parallel and one antiparallel β-strands. The large domain is similar to the small domain but contains two additional α-helices and three more short antiparallel β-strands. A comparison of the secondary structural motifs of GlnBP with those of other periplasmic binding proteins is discussed.A model of the “closed form” GlnBP-Gln complex has been proposed based on the crystal structures of the histidine-binding protein-His complex and “open form” GlnBP. This model has been successfully used as a search model in the crystal structure determination of the “closed form” GlnBP-Gln complex by molecular replacement methods. The model agrees remarkably well with the crystal structure of the Gln-GlnBP complex with root-mean-square deviation of 1.29 Å. Our study shows that, at least in our case, it is possible to predict one conformational state of a periplasmic binding protein from another conformational state of the protein. The glutamine-binding pockets of the model and the crystal structure are compared and the modeling technique is described.     

Crystal structures of the tryptophan repressor binding protein WrbA and complexes with flavin mononucleotide

The tryptophan repressor binding protein WrbA binds to the tryptophan repressor protein TrpR. Although the biological role of WrbA remains unclear, it has been proposed to function in enhancing the stability of TrpR-DNA complexes. Sequence database analysis has identified WrbA as a founding member of a flavodoxin-like family of proteins. Here we present crystal structures of WrbA from Deinococcus radiodurans and Pseudomonas aeruginosa and their complexes with flavin mononucleotide. The protomer structure is similar to that of previously determined long-chain flavodoxins; however, each contains a conserved inserted region unique to the WrbA family. Interestingly, each WrbA protein forms a homotetramer with 222 symmetry, unique among flavodoxin-like proteins, in which each protomer binds one flavin mononucleotide cofactor molecule.     

Structural and functional consequences of methionine oxidation in thrombomodulin

Thrombomodulin (TM) is an endothelial cell surface glycoprotein that is responsible for switching the catalytic activity of thrombin away from fibrinogen cleavage (pro-coagulant) and towards protein C cleavage (anticoagulant). Although TM is a large protein, only the fourth and fifth epidermal growth factor-like (EGF-like) domains are required for anticoagulant function. These two domains must work together, and the linker between the two domains contains a single methionine residue, Met 388. Oxidation of Met 388 is deleterious for TM activity. Structural studies, both X-ray and NMR, of wild type and variants at position 388 show that Met 388 provides a key linkage between the two domains. Oxidation of the methionine has consequences for the structure of the fifth domain, which binds to thrombin. Oxidation also appears to disrupt the interdomain contacts resulting in structural and dynamic changes. The functional consequences of oxidation of Met 388 include decreased anticoagulant activity. Oxidative stress from several causes is reflected in lower serum levels of activated protein C and a higher thrombotic tendency, and this is thought to be linked to the oxidation of Met 388 in TM. Thus, TM structure and function are altered in a subtle but functionally critical way upon oxidation of Met 388.     

Crystal structure of TM1457 from Thermotoga maritima

The crystal structure of a hypothetical protein, TM1457, from Thermotoga maritima has been determined at 2.0A resolution. TM1457 belongs to the DUF464 family (57 members) for which there is no known function. The structure shows that it is composed of two helices in contact with one side of a five-stranded beta-sheet. Two identical monomers form a pseudo-dimer in the asymmetric unit. There is a large cleft between the first alpha-helix and the second beta-strand. This cleft may be functionally important, since the two highly conserved motifs, GHA and VCAXV(S/T), are located around the cleft. A structural comparison of TM1457 with known protein structures shows the best hit with another hypothetical protein, Ybl001C from Saccharomyces cerevisiae, though they share low structural similarity. Therefore, TM1457 still retains a unique topology and reveals a novel fold.