Xanthine nucleotide binding Go alpha mutant

B. Yu, V. Slepak, M. I. Simon

Research output: Contribution to journalArticle

Abstract

To better understand the G-protein coupled signal transduction network and to activate a particular Go in vivo without interference with other G proteins, we switched the nucleotide -binding specificity of Goa from guanine to a non-natural ligand, xanthine nucleotide, by site-directed mutagenesis. Crystal structures of a subunits of transducin and Gi shows that a conserved aspartic acid in the NKXD motif forms hydrogen bonds to the Nl nitrogen and the N2 amine of the bound guanine ring. We changed this aspartic acid (Asp273) of Goa to asparagine which may coordinate to xanthine instead of guanine. This mutation was introduced in the wild type Goa subunit and the GTPase-deficient Goα mutant (Q205L). Both GoαDN and the double mutant GoαDNQL were expressed in E.coli and purified. GoαDNQL showed strong affinity to XTPγS, but not to GTPγS. Interestingly, only the double mutant was active, while GoαDN did not bind either GTPγS or XTPγS. Recombinant Goa mutants were further characterized by limited proteolysis with trypsin and by pertussis toxin induced ADP-ribosylation. GoαDN was found not active in both assays. GoαDNQL showed similar functions like wild type Goα, but was regulated by xanthine nucleotides rather than guanine nucleotides. GoαDNQL formed complex with βγ subunits only in the presence of XDP, and the trimerization was inhibited by XTP. After binding XTP, GoαDNQL underwent the characteristic conformational change associated with wild type G-proteins, indicating GoαDNQL was activated by XTP.

Original languageEnglish (US)
Pages (from-to)A1261
JournalFASEB Journal
Volume10
Issue number6
StatePublished - Dec 1 1996
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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  • Cite this

    Yu, B., Slepak, V., & Simon, M. I. (1996). Xanthine nucleotide binding Go alpha mutant. FASEB Journal, 10(6), A1261.