Specific and effective interaction of a guanine nucleotide analogue with small G proteins

Simon Hoffenberg, Timothy M. Shannon, Timothy P. Noonan, Shaobin Liu, D. Sundarsingh Daniel, Jordan B. Fishman, Jeffrey B. Rubins, Hemant K. Misra, George E. Wright, Burton F. Dickey

Research output: Contribution to journalArticle

6 Scopus citations

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 [α-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'-(β,γ-difluoromethylene)triphosphate, had a ~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 β,γ- 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'-(β,γ- 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'-(β,γ- difluoromethylene)triphosphate protected a 20-kDa fragment of rab5 from tryptic proteolysis with greater efficacy than guanosine-5'-O-(3- thiotriphosphate) or guanosine-5'-(β,γ-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.

Original languageEnglish (US)
Pages (from-to)156-164
Number of pages9
JournalMolecular Pharmacology
Volume49
Issue number1
StatePublished - Jan 1996

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    Hoffenberg, S., Shannon, T. M., Noonan, T. P., Liu, S., Daniel, D. S., Fishman, J. B., Rubins, J. B., Misra, H. K., Wright, G. E., & Dickey, B. F. (1996). Specific and effective interaction of a guanine nucleotide analogue with small G proteins. Molecular Pharmacology, 49(1), 156-164.