Specific and effective interaction of a guanine nucleotide analogue with small G proteins.
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.
Hoffenberg
S
S
Department of Medicine, Boston University, School of Medicine, Massachusetts, USA.
Shannon
T M
TM
Noonan
T P
TP
Liu
S
S
Daniel
D S
DS
Fishman
J B
JB
Rubins
J B
JB
Misra
H K
HK
Wright
G E
GE
Dickey
B F
BF
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
D006150:Guanine Nucleotides; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins