Evidence for two distinct Mg 2+ binding sites in G sα and G iα1 proteins

Abstract

The function of guanine nucleotide binding (G) proteins is Mg 2+ dependent with guanine nucleotide exchange requiring higher metal ion concentration than guanosine 5′-triphosphate hydrolysis. It is unclear whether two Mg 2+ binding sites are present or if one Mg 2+ binding site exhibits different affinities for the inactive GDP-bound or the active GTP-bound conformations. We used furaptra, a Mg 2+-specific fluorophore, to investigate Mg 2+ binding to α subunits in both conformations of the stimulatory (G sα) and inhibitory (G iα1) regulators of adenylyl cyclase. Regardless of the conformation or α protein studied, we found that two distinct Mg 2+ sites were present with dissimilar affinities. With the exception of G sα in the active conformation, cooperativity between the two Mg 2+ sites was also observed. Whereas the high affinity Mg 2+ site corresponds to that observed in published X-ray structures of G proteins, the low affinity Mg 2+ site may involve coordination to the terminal phosphate of the nucleotide.