Evidence for two distinct Mg2+ binding sites in G(s alpha) and G(i alpha1) 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 alpha subunits in both conformations of the stimulatory (G(s alpha)) and inhibitory (G(i alpha1)) regulators of adenylyl cyclase. Regardless of the conformation or alpha protein studied, we found that two distinct Mg(2+) sites were present with dissimilar affinities. With the exception of G(s alpha) 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.