title: Characterization of the second ion-binding site in the G domain of H-Ras.
abstract: Ras is a small monomeric GTPase acting as molecular switch in multiple cellular processes. The N-terminal G domain of Ras binds GTP or GDP accompanied by a magnesium ion, which is strictly required for GTPase activity and performs a structural role. Another ion-binding site on the opposite face of the G domain has been recently observed to specifically associate with calcium acetate in the crystal [Buhrman, G., et al. (2010) Proc. Natl. Aacd. Sci. U.S.A. 107, 4931-4936]. In this article, we report thermodynamic measurements of the affinity and specificity of the remote ion-binding site in H-Ras as observed in solution. Using (15)N-(1)H nuclear magnetic resonance spectroscopy, we determined that, in contrast to the crystalline state, the remote site in solution is specific for a divalent cation, binding both calcium and magnesium with anions playing a minimal role. The affinity of the remote site for divalent cations is in the low millimolar range and remarkably different for GDP- and GppNHp-bound forms of the G domain, indicating that the GTP-binding pocket and the remote site are allosterically coupled through the distance of more than 25 Å. Considering that the remote site is oriented toward the membrane surface in vivo, we hypothesize that its cognate biological ligand might be a positively charged group extending from a lipid or an integral membrane protein.
authors.affiliation: Biochemistry Department, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
authors.forename: Casey
authors.initials: C
authors.lastname: O'Connor
authors.forename: Evgenii L
authors.initials: EL
authors.lastname: Kovrigin
mesh_terms: D001665:Binding Sites; D002413:Cations, Divalent; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D015689:Oncogene Protein p21(ras) / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013816:Thermodynamics
chemical_list: D002413:Cations, Divalent; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D015689:Oncogene Protein p21(ras)