TY - JOUR
T1 - Specific and effective interaction of a guanine nucleotide analogue with small G proteins
AU - Hoffenberg, Simon
AU - Shannon, Timothy M.
AU - Noonan, Timothy P.
AU - Liu, Shaobin
AU - Daniel, D. Sundarsingh
AU - Fishman, Jordan B.
AU - Rubins, Jeffrey B.
AU - Misra, Hemant K.
AU - Wright, George E.
AU - Dickey, Burton F.
PY - 1996/1
Y1 - 1996/1
N2 - 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.
AB - 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.
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M3 - Article
C2 - 8569702
AN - SCOPUS:9044239668
SN - 0026-895X
VL - 49
SP - 156
EP - 164
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 1
ER -