Evidence for two distinct Mg2+ binding sites in G(s alpha) and G(i alpha1) proteins.
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.
Department of Chemistry, Loyola University Chicago, 1068 W. Sheridan Road, Flanner Hall, Chicago, IL 60626, USA.
Christopher S
CS
Malarkey
Guoyan
G
Wang
Miguel A
MA
Ballicora
Duarte E
DE
Mota de Freitas
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D001665:Binding Sites; D005456:Fluorescent Dyes / Q000737:chemistry; D016257:Fura-2 / Q000031:analogs & derivatives / Q000737:chemistry; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation
D000067956:Adenylyl Cyclase Inhibitors; D005456:Fluorescent Dyes; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; D008274:Magnesium; D016257:Fura-2