A novel signal transduction protein: Combination of solute binding and tandem PAS-like sensor domains in one polypeptide chain.

We report the structural and biochemical characterization of a novel periplasmic ligand-binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from Lake Retba, in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N-terminal and a tandem PAS-like sensor domain at the C-terminal region. SBP domains are found ubiquitously, and their best known function is in solute transport across membranes. PAS-like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes but have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS-like domain of the tandem PAS-like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS-like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non-redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport.

Structural studies of ROK fructokinase YdhR from Bacillus subtilis: insights into substrate binding and fructose specificity.

The main pathway of bacterial sugar phosphorylation utilizes specific phosphoenolpyruvate phosphotransferase system (PTS) enzymes. In addition to the classic PTS system, a PTS-independent secondary system has been described in which nucleotide-dependent sugar kinases are used for monosaccharide phosphorylation. Fructokinase (FK), which phosphorylates d-fructose with ATP as a cofactor, has been shown to be a member of this secondary system. Bioinformatic analysis has shown that FK is a member of the "ROK" (bacterial Repressors, uncharacterized Open reading frames, and sugar Kinases) sequence family. In this study, we report the crystal structures of ROK FK from Bacillus subtilis (YdhR) (a) apo and in the presence of (b) ADP and (c) ADP/d-fructose. All structures show that YdhR is a homodimer with a monomer composed of two similar α/ß domains forming a large cleft between domains that bind ADP and D-fructose. Enzymatic activity assays support YdhR function as an ATP-dependent fructose kinase.

Sequence of the Octopus dofleini hemocyanin subunit: structural and evolutionary implications.

Sequencing of the subunit of the hemocyanin of Octopus dofleini has been completed from a cDNA library. This represents the first molluscan hemocyanin to be completely sequenced. The sequence determined is for one of the two distinguishable cDNAs which have been recognized for this protein. The protein subunit has 2896 amino acids and contains seven functional units, each carrying two sets of three invariant histidine residues constituting the binding sites (A and B) for two copper atoms. The accompanying paper identifies this site in the C-terminal functional unit (Odg). Differences in sequence for the two cDNAs, for the region in which both are available, are concentrated in the "linker regions" between functional units. The sequences of the seven units exhibit high similarity, averaging about 40% identity, with a concentration of conserved sequences in the region surrounding the copper binding sites. The sequences around the B-site show significant homology to the sequences of arthropod hemocyanins. Comparison of the functional unit sequences in terms of hydrophobicity and surface exposure profiles, as well as regions of probable secondary structure, indicate that all functional units probably have a common tertiary folding; the protein subunit is a string of similarly folded beads. A number of putative N-linked carbohydrate binding sites can be recognized in the sequence; one of these corresponds to the carbohydrate observed in the X-ray diffraction study of functional unit Odg as disclosed in the accompying paper. Phylogenetic analysis of the sequences of the O. dofleini functional units, and comparison with other available molluscan sequences indicates that the multi-domain subunit structure must have arisen over a relatively brief period, preceeding the differentiation of major molluscan types.

Crystal structure of a functional unit from Octopus hemocyanin.

Hemocyanins are giant oxygen transport proteins found in many arthropods and molluscs. Freely dissolved in the hemolymph, they are multisubunit proteins that contain many copies of the active site, a copper atom pair that reversibly binds oxygen. Octopus hemocyanin is composed of ten subunits, each of which contain seven oxygen-binding "functional units". The carboxyl-terminal 47 kDa functional unit, Odg, is a proteolytic isolate that binds oxygen reversibly while exhibiting slight Bohr and magnesium ion effects. In this work we present the X-ray structure determination and analysis of Odg at 2.3 A resolution. Odg has two structural domains: a largely alpha-helical copper binding domain, and a five-stranded anti-parallel beta-sandwich with the jelly roll topology found in many viruses. Six histidine residues ligate the copper atoms, one of which is involved in a thioether bridge. The results show that the hemocyanin from the mollusc and that from the arthropod have distinct tertiary folds in addition to the long recognized differences in their quaternary structures. Nonetheless, a comparison of Octopus and horseshoe crab hemocyanin reveals a similar active site, in a striking example of perhaps both convergent and divergent evolution.

Crystals of the carboxyl-terminal functional unit from Octopus dofleini hemocyanin.

The carboxyl-terminal oxygen-binding unit of the polypeptide from Octopus dofleini hemocyanin has been crystallized in a form suitable for three-dimensional X-ray analysis. This proteolytic fragment has a molecular weight of 47 kDa and reversibly binds O2 while exhibiting a slight Bohr effect. Two types of crystals have been grown. Type I crystals, currently under analysis, belong to the orthorhombic space group P2(1)2(1)2(1) and have unit cell dimensions of 92.6 A x 167.4 A x 59.2 A. A composition of two protein molecules per asymmetric unit and 50% solvent content is consistent with a self-rotation function that identifies a non-crystallographic 2-fold axis of symmetry relating these molecules. Diffraction extending beyond 1.9 A Bragg spacings can be detected with synchrotron X-radiation.

Crystallization and characterization of human chorionic gonadotropin in chemically deglycosylated and enzymatically desialylated states.

Crystals suitable for X-ray diffraction studies at moderate resolution have been grown from two forms of human chorionic gonadotropin (hCG): HF-treated hCG and neuraminidase-treated hCG. The enzymatically desialylated form of hCG produced crystals that diffract to 2.8 A as compared to the HF-treated hCG crystals that diffract to 3.0 A. Although it was assumed that the high and heterogeneous carbohydrate content of the glycoprotein hormones inhibited their crystallization, this report suggests that it is the negatively charged surface sugars and neither the total carbohydrate content nor its heterogeneity which interferes with crystal formation. Chemical deglycosylation resulted in significantly increased protein degradation during crystal growth. Such peptide bond cleavages were observed to a much lesser extent in the crystals grown from neuraminidase-digested hCG. Sequence analysis of the HF-treated hCG crystals suggested that up to 45% of the molecules within the crystal had an acid-labile peptide bond cleaved. In contrast, the neuraminidase-treated hCG exhibited less than 9% of this type of cleavage. The increase in heterogeneity of the polypeptide chains within both crystals over that existent in the starting proteins was apparently due to changes occurring during crystal growth. The manner in which hCG was treated prior to crystallization was found to be a very important factor in the extent of peptide bond cleavages occurring during crystal growth. HF treatment of glycoproteins may render glycoproteins more susceptible to peptide bond cleavages during crystal growth.