Crystal structure of muconate lactonizing enzyme at 3 A resolution

The crystal structure of muconate lactonizing enzyme has been solved at 3 A resolution, and an unambiguous alpha-carbon backbone chain trace made. The enzyme contains three domains; the central domain is a parallel-stranded alpha-beta barrel, which has previously been reported in six other enzymes, including triose phosphate isomerase and pyruvate kinase. One novel feature of this enzyme is that its alpha-beta barrel has only seven parallel alpha-helices around the central core of eight parallel beta-strands; all other known alpha-beta barrels contain eight such helices. The N-terminal (alpha + beta) and C-terminal domains cover the cleft where the eighth helix would be. The active site of muconate lactonizing enzyme has been found by locating the manganese ion that is essential for catalytic activity, and by binding and locating an inhibitor, alpha-ketoglutarate. The active site lies in a cleft between the N-terminal and barrel domains; when the active sites of muconate lactonizing enzyme and triose phosphate isomerase are superimposed, barrel-strand 1 of triose phosphate isomerase is aligned with barrel-strand 3 of muconate lactonizing enzyme. This implies that structurally homologous active-site residues in the two enzymes are carried on different parts of the primary sequence; the ancestral gene would had to have been transposed during its evolution to the modern proteins, which seems unlikely. Therefore, these two enzymes may be related by convergent, rather than divergent, evolution.     

Electrostatic calculations and model-building suggest that DNA bound to CAP is sharply bent

Two observations suggest that DNA, upon binding to E. coli catabolite gene activator protein (CAP), is sharply bent by a total angle of at least 100-150 degrees: (1) The electrostatic potential field of CAP shows regions of positive potential that form a ramp on 3 sides of the protein. (2) The DNA binding site size as determined by DNA ethylation interference with binding, (Majors: "Control of the E. coli Lac Operon at the Molecular Level." Ph.D. Thesis, Harvard University, Cambridge, 1977) and by relative affinities of DNA fragments of various lengths (Liu-Johnson et al.: Cell 47:995-1005, 1986) requires severe bending of the DNA to maintain its favorable electrostatic contact with the protein.     

The cellular RNA-binding protein EAP recognizes a conserved stem-loop in the Epstein-Barr virus small RNA EBER 1.

EAP (EBER-associated protein) is an abundant, 15-kDa cellular RNA-binding protein which associates with certain herpesvirus small RNAs. We have raised polyclonal anti-EAP antibodies against a glutathione S-transferase-EAP fusion protein. Analysis of the RNA precipitated by these antibodies from Epstein-Barr virus (EBV)- or herpesvirus papio (HVP)-infected cells shows that > 95% of EBER 1 (EBV-encoded RNA 1) and the majority of HVP 1 (an HVP small RNA homologous to EBER 1) are associated with EAP. RNase protection experiments performed on native EBER 1 particles with affinity-purified anti-EAP antibodies demonstrate that EAP binds a stem-loop structure (stem-loop 3) of EBER 1. Since bacterially expressed glutathione S-transferase-EAP fusion protein binds EBER 1, we conclude that EAP binding is independent of any other cellular or viral protein. Detailed mutational analyses of stem-loop 3 suggest that EAP recognizes the majority of the nucleotides in this hairpin, interacting with both single-stranded and double-stranded regions in a sequence-specific manner. Binding studies utilizing EBER 1 deletion mutants suggest that there may also be a second, weaker EAP-binding site on stem-loop 4 of EBER 1. These data and the fact that stem-loop 3 represents the most highly conserved region between EBER 1 and HVP 1 suggest that EAP binding is a critical aspect of EBER 1 and HVP 1 function.     

WNT-mediated relocalization of dishevelled proteins

Summary TheWnt family of proto-oncogenes encodes secreted signaling proteins that are required for mouse development. TheDrosophila Wnt homolog, thewingless (Wg) segment polarity gene, mediates a signal transduction pathway in which the downstream elements appear to be conserved through evolution. One such element, thedishevelled gene product, becomes hyperphosphorylated and translocates to the plasma membrane in response to Wg (Yanagawa et al., 1995). We report here that the mouseDishevelle-1 (Dvl-1) andDishevelled-2 genes encode proteins that are differentially localized inWnt-overexpressing PC12 cell lines (PC12/Wnt). WhereasDvl-1 andDvl-2 proteins are limited to the soluble fraction of parental PC12 cells, PC12/Wnt cells display a subset ofDvl-1 protein associated with the membrane andDvl-2 protein with the cytoskeletal fraction. These results suggest a conserved role forDvl inWnt/wg signal transduction.     

High resolution X-ray structure of yeast hexokinase,an allosteric protein exhibiting a non-symmetric arrangement of subunits

The structure determination of yeast hexokinase has been extended to 3.5 Å resolution for the dimer and to 2.7 Å resolution for the monomer using multiple isomorphous replacement. The electron density maps of both the monomer and dimer crystal forms have been substantially improved by an averaging procedure. From these maps the course of the polypeptide backbone and some aspects of the dimer interaction have been established.The hexokinase subunit arrangement is contrary to a major tenet of the Monod et al. (1965) theory of allosteric proteins which postulated that only symmetric or isologous interactions of subunits would occur in oligomeric proteins. One subunit of the dimer is related to the other by a 156 ° rotation about and a 13.8 Å translation along a molecular screw axis. In the hexokinase dimer the set of residues in one subunit that is interacting with the other subunit is different from the set of residues in the second subunit that is interacting with the first subunit. This heterologous or non-symmetric interaction of subunits is associated with some small differences in the structure of the two subunits, particularly at the subunit interface, and accounts for some of this enzyme's non-symmetric interactions with substrates and activators. Indeed, the non-symmetric subunit association may play an important role in the control of this enzyme's activity.The overall structure of hexokinase is considerably different than the known structures of the other enzymes in the glycolytic pathway. Although there is a striking similarity between the domain of hexokinase that binds AMP and the domain of lactate dehydrogenase that binds NAD, the former structure contains both antiparallel and parallel β-pleated strands, while the latter contains only parallel β-structure. In an attempt to assess the significance of this structural similarity, the structure of the nucleotide binding domains of hexokinase and lactate dehydrogenase are compared to a portion of carboxypeptidase A. The observed similarities among these structures suggests that a central β-pleated sheet flanked by α-helices is a common supersecondary structure that probably arose by convergent as well as divergent evolution. Thus, there appears to be no compelling evidence at this time to support the hypothesis that a part of hexokinase has evolved from the same gene as the dinucleotide binding domain of lactate dehydrogenase.     

Structure of yeast hexokinase: IV. Low-resolution structure of enzyme—Substrate complexes revealing negative co-operativity and allosteric interactions

We conclude from X-ray diffraction studies at low resolution (7 Å) that the binding of sugar and nucleotide substrates to dimeric yeast hexokinase BII crystals exhibits both negative co-operativity and positive allosteric co-operativity. Difference electron density maps show the positions of sugar and nucleotide binding sites and extensive substrate-induced structural changes in the protein. Sugar substrates and inhibitors bind in the deep cleft that divides each subunit into two lobes and nucleotide substrates bind nearby to one site per dimer, which lies between the subunits and on the molecular symmetry axis. Although the inhibitors o- and p-iodobenzoylglucosamine and o-toluoylglucosamine bind equally to both subunits, the degree of substitution of glucose or xylose is very different for the two subunits. The substrate analog β, γ-imido ATP shows only one strong binding site per dimer. This negative co-operativity in substrate binding may result from the heterologous or non-equivalent association of the two subunits (Anderson et al., 1974), which provides non-equivalent environments for the two chemically identical subunits.Further, there is a positive allosteric interaction between the sugar and nucleotide binding sites. Sugar binding is required for nucleotide binding at the intersubunit site and the binding of nucleotide modifies the binding of sugars. These positive heterotropic interactions appear to be mediated by extensive substrate-induced structural changes in the enzyme.