Magnesium fluoride-dependent binding of small G proteins to their GTPase-activating proteins.
GTPase-activating proteins (GAPs) enhance the intrinsic GTPase activity of small G proteins, such as Ras and Rho, by contributing a catalytic arginine to the active site. An intramolecular arginine plays a similar role in heterotrimeric G proteins. Aluminum fluoride activates the GDP form of heterotrimeric G proteins, and enhances binding of the GDP form of small G proteins to their GAPs. The resultant complexes have been interpreted as analogues of the transition state of the hydrolytic reaction. Here, equilibrium binding has been measured using scintillation proximity assays to provide quantitative information on the fluoride-mediated interaction of Ras and Rho proteins with their respective GAPs, neurofibromin (NF1) and RhoGAP. High-affinity fluoride-mediated complex formation between Rho.GDP and RhoGAP occurred in the absence of aluminum; however, under these conditions, magnesium was required. Additionally, the novel observation was made of magnesium-dependent, fluoride-mediated binding of Ras.GDP to NF1 in the absence of aluminum. Aluminum was required for complex formation when the concentration of magnesium was low. Thus, either aluminum fluoride or magnesium fluoride can mediate the high-affinity binding of Rho. GDP or Ras.GDP to GAPs. It has been reported that magnesium fluoride can activate heterotrimeric G proteins. Thus, magnesium-dependent fluoride effects might be a general phenomenon with G proteins. Moreover, these data suggest that some protein.nucleotide complexes previously reported to contain aluminum fluoride may in fact contain magnesium fluoride.
Graham
D L
DL
Division of Physical Biochemistry, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
Eccleston
J F
JF
Chung
C W
CW
Lowe
P N
PN
D000535:Aluminum / Q000378:metabolism; D005459:Fluorides / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D017616:Magnesium Compounds / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D025542:Neurofibromin 1; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism; D012969:Sodium Fluoride / Q000494:pharmacology; D014961:X-Ray Diffraction; D018631:ras Proteins / Q000378:metabolism; D020743:rhoB GTP-Binding Protein / Q000378:metabolism
D020690:GTPase-Activating Proteins; D017616:Magnesium Compounds; D025542:Neurofibromin 1; D011506:Proteins; C085379:rho GTPase-activating protein; D015636:Magnesium Chloride; D006153:Guanosine Diphosphate; C031288:magnesium fluoride; D012969:Sodium Fluoride; D000535:Aluminum; D020559:Monomeric GTP-Binding Proteins; D018631:ras Proteins; D020743:rhoB GTP-Binding Protein; D005459:Fluorides