title: GDP affinity and order state of the catalytic site are critical for function of xanthine nucleotide-selective Galphas proteins.
abstract: Xanthine nucleotide-selective small GTP-binding proteins with an Asp/Asn mutation are valuable for the analysis of individual GTP-binding proteins in complex systems. Similar applications can be devised for heterotrimeric G-proteins. However, Asp/Asn mutants of Galpha(o), Galpha(11), and Galpha(16) were inactive. An additional Gln/Leu mutation in the catalytic site, reducing GTPase activity and increasing GDP affinity, was required to generate xanthine nucleotide-selective unspecified G-protein alpha-subunit (Galpha). Our study aim was to generate xanthine nucleotide-selective mutants of Galpha(s), the stimulatory G-protein of adenylyl cyclase. The short splice variant of Galpha(s) (Galpha(sS)) possesses higher GDP affinity than the long splice variant (Galpha(sL)). Nucleoside 5'-[gamma-thio]triphosphates (NTPgammaSs) and nucleoside 5'-[beta,gamma-imido]triphosphates effectively activated a Galpha(sS) mutant with a D280N exchange (Galpha(sS)-N280), whereas nucleotides activated a Galpha(sL) mutant with a D295N exchange (Galpha(sL)-N295) only weakly. The Gln/Leu mutation enhanced Galpha(sL)-N295 activity. NTPgammaSs activated Galpha(sS)-N280 and a Galpha(sL) mutant with a Q227L and D295N exchange (Galpha(sL)-L227/N295) with similar potencies, whereas xanthosine 5'-triphosphate and xanthosine 5'-[beta,gamma-imido]triphosphate were more potent than GTP and guanosine 5'-[beta,gamma-imido]triphosphate, respectively. Galpha(sS)-N280 interacted with the beta(2)-adrenoreceptor and exhibited high-affinity XTPase activity. Collectively, (i) Galpha(sS)-N280 is the first functional xanthine nucleotide-selective Galpha with the Asp/Asn mutation alone; (ii) sufficiently high GDP affinity is crucial for Galpha Asp/Asn mutant function; (iii) with nucleoside 5'-triphosphates and nucleoside 5'-[beta,gamma-imido]triphosphates, Galpha(s)-N280 and Galpha(sL)-L227/N295 exhibit xanthine nucleotide selectivity, whereas NTPgammaSs sterically perturb the catalytic site of Galpha and annihilate xanthine selectivity.
authors.affiliation: Department of Pharmacology and Toxicology, The University of Kansas, Lawrence 66045-7582, USA.
authors.forename: Andreas
authors.initials: A
authors.lastname: Gille
authors.forename: Katharina
authors.initials: K
authors.lastname: Wenzel-Seifert
authors.forename: Michael B
authors.initials: MB
authors.lastname: Doughty
authors.forename: Roland
authors.initials: R
authors.lastname: Seifert
mesh_terms: D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism / Q000502:physiology*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D010766:Phosphorylation; D018411:Spodoptera; D019820:Xanthine / Q000378:metabolism*
chemical_list: D009711:Nucleotides; D006153:Guanosine Diphosphate; D019820:Xanthine; D019205:GTP-Binding Protein alpha Subunits, Gs