title: Specific and effective interaction of a guanine nucleotide analogue with small G proteins.
abstract: G proteins are molecular switches that use a cycle of GTP binding and hydrolysis to regulate a wide variety of cellular biochemical processes. Because the functional state of these proteins is allosterically determined by bound guanine nucleotides, a nucleotide analogue with protein specificity might have pharmacological or biochemical value. The binding of [alpha-32P]GTP to four small G proteins immobilized on nitrocellulose was competed by a series of analogues with modifications at multiple sites. One analogue, N2-(p-n-butylphenyl)guanosine 5'-(beta,gamma-difluoromethylene)triphosphate, had a approximately 40-fold higher affinity for one small G protein than for two of the others. Systematic analysis of each modification in the synthetic nucleotide revealed that specificity was conferred by the carbon substitution in the beta,gamma-phosphoanhydride bond. These observations were then extended to purified proteins of known sequence in solution by filtration binding studies with H-ras and rab5. Ras was 9-fold more discriminant between guanosine-5'-(beta,gamma-difluoromethylene)triphosphate and guanosine-5'-O-(3-thiotriphosphate) than was rab5, and the Q79L GTPase-defective mutant of rab5 was 6-fold more discriminant than wild-type rab5. Guanosine-5'-(beta,gamma-difluoromethylene)triphosphate protected a 20-kDa fragment of rab5 from tryptic proteolysis with greater efficacy than guanosine-5'-O-(3-thiotriphosphate) or guanosine-5'-(beta,gamma-imido)triphosphate despite its lower affinity, and GMP stabilized a conformation indistinguishable from apo-rab5. These results identify a synthetic guanine nucleotide analogue with differential affinity for closely related G proteins, determine the atomic substitution in the analogue that confers specificity, demonstrate discrimination by the analogue between wild-type and a point-mutant G protein, and establish efficacy of the analogue in inducing conformational change of a target protein disproportionate to the affinity of the interaction.
authors.affiliation: Department of Medicine, Boston University, School of Medicine, Massachusetts, USA.
authors.forename: S
authors.initials: S
authors.lastname: Hoffenberg
authors.forename: T M
authors.initials: TM
authors.lastname: Shannon
authors.forename: T P
authors.initials: TP
authors.lastname: Noonan
authors.forename: S
authors.initials: S
authors.lastname: Liu
authors.forename: D S
authors.initials: DS
authors.lastname: Daniel
authors.forename: J B
authors.initials: JB
authors.lastname: Fishman
authors.forename: J B
authors.initials: JB
authors.lastname: Rubins
authors.forename: H K
authors.initials: HK
authors.lastname: Misra
authors.forename: G E
authors.initials: GE
authors.lastname: Wright
authors.forename: B F
authors.initials: BF
authors.lastname: Dickey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D002417:Cattle; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D006150:Guanine Nucleotides; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins