title: Crystal Structure of Recoverin with Calcium Ions Bound to Both Functional EF Hands.
abstract: Recoverin (Rv), a small Ca(2+)-binding protein that inhibits rhodopsin kinase (RK), has four EF hands, two of which are functional (EF2 and EF3). Activation requires Ca(2+) in both EF hands, but crystal structures have never been observed with Ca(2+) ions in both sites; all previous structures have Ca(2+) bound to only EF3. We suspected that this was due to an intermolecular crystal contact between T80 and a surface glutamate (E153) that precluded coordination of a Ca(2+) ion in EF2. We constructed the E153A mutant, determined its X-ray crystal structure to 1.2 Å resolution, and showed that two Ca(2+) ions are bound, one in EF3 and one in EF2. Additionally, several other residues are shown to adopt conformations in the 2Ca(2+) structure not seen previously and not seen in a second structure of the E153A mutant containing Na(+) instead of Ca(2+) in the EF2 site. The side-chain rearrangements in these residues form a 28 Å allosteric cascade along the surface of the protein connecting the Ca(2+)-binding site of EF2 with the active-site pocket responsible for binding RK.
authors.affiliation: Department of Biochemistry, Brandeis University , Waltham, Massachusetts 02454, United States.
authors.forename: Ramasamy P
authors.initials: RP
authors.lastname: Kumar
authors.affiliation: Department of Biochemistry, Brandeis University , Waltham, Massachusetts 02454, United States.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Ranaghan
authors.affiliation: Department of Biochemistry, Brandeis University , Waltham, Massachusetts 02454, United States.
authors.forename: Allen Y
authors.initials: AY
authors.lastname: Ganjei
authors.affiliation: Department of Biochemistry, Brandeis University , Waltham, Massachusetts 02454, United States.
authors.forename: Daniel D
authors.initials: DD
authors.lastname: Oprian
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002413:Cations, Divalent / Q000737:chemistry; D018360:Crystallography, X-Ray; D006801:Humans; D020125:Mutation, Missense; D051603:Recoverin / Q000737:chemistry* / Q000235:genetics
chemical_list: D002413:Cations, Divalent; C496731:RCVRN protein, human; D051603:Recoverin; D002118:Calcium