title: Characterization of a Goalpha mutant that binds xanthine nucleotides.
abstract: Several GTP binding proteins, including EF-Tu, Ypt1, rab-5, and FtsY, and adenylosuccinate synthetase have been reported to bind xanthine nucleotides when the conserved aspartate residue in the NKXD motif was changed to asparagine. However, the corresponding single Goalpha mutant protein (D273N) did not bind either xanthine nucleotides or guanine nucleotides. Interestingly, the introduction of a second mutation to generate the Goalpha subunit D273N/Q205L switched nucleotide binding specificity to xanthine nucleotide. The double mutant protein GoalphaD273N/Q205L (GoalphaX) bound xanthine triphosphate, but not guanine triphosphate. Recombinant GoalphaX (GoalphaD273N/Q205L) formed heterotrimers with betagamma complexes only in the presence of xanthine diphosphate (XDP), and the binding to betagamma was inhibited by xanthine triphosphate (XTP). Furthermore, as a result of binding to XTP, the GoalphaX protein underwent a conformational change similar to that of the activated wild-type Goalpha. In transfected COS-7 cells, we demonstrate that the interaction between GoalphaX and betagamma occurred only when cell membranes were permeabilized to allow the uptake of xanthine diphosphate. This is the first example of a switch in nucleotide binding specificity from guanine to xanthine nucleotides in a heterotrimeric G protein alpha subunit.
authors.affiliation: Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: B
authors.initials: B
authors.lastname: Yu
authors.forename: V Z
authors.initials: VZ
authors.lastname: Slepak
authors.forename: M I
authors.initials: MI
authors.lastname: Simon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D006150:Guanine Nucleotides; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012265:Ribonucleotides; D010566:Virulence Factors, Bordetella; D009243:NAD; C065326:xanthosine 5'-triphosphate; D019204:GTP-Binding Proteins