title: Designing sequence to control protein function in an EF-hand protein.
abstract: The extent of conformational change that calcium binding induces in EF-hand proteins is a key biochemical property specifying Ca(2+) sensor versus signal modulator function. To understand how differences in amino acid sequence lead to differences in the response to Ca(2+) binding, comparative analyses of sequence and structures, combined with model building, were used to develop hypotheses about which amino acid residues control Ca(2+)-induced conformational changes. These results were used to generate a first design of calbindomodulin (CBM-1), a calbindin D(9k) re-engineered with 15 mutations to respond to Ca(2+) binding with a conformational change similar to that of calmodulin. The gene for CBM-1 was synthesized, and the protein was expressed and purified. Remarkably, this protein did not exhibit any non-native-like molten globule properties despite the large number of mutations and the nonconservative nature of some of them. Ca(2+)-induced changes in CD intensity and in the binding of the hydrophobic probe, ANS, implied that CBM-1 does undergo Ca(2+) sensorlike conformational changes. The X-ray crystal structure of Ca(2+)-CBM-1 determined at 1.44 A resolution reveals the anticipated increase in hydrophobic surface area relative to the wild-type protein. A nascent calmodulin-like hydrophobic docking surface was also found, though it is occluded by the inter-EF-hand loop. The results from this first calbindomodulin design are discussed in terms of progress toward understanding the relationships between amino acid sequence, protein structure, and protein function for EF-hand CaBPs, as well as the additional mutations for the next CBM design.
authors.affiliation: Department of Biochemistry, Center for Structural Biology, Vanderbilt University, 5140 BIOSCI/MRB III, Nashville, Tennessee 37232-8725, USA.
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Bunick
authors.forename: Melanie R
authors.initials: MR
authors.lastname: Nelson
authors.forename: Sheryll
authors.initials: S
authors.lastname: Mangahas
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Hunter
authors.forename: Jonathan H
authors.initials: JH
authors.lastname: Sheehan
authors.forename: Laura S
authors.initials: LS
authors.lastname: Mizoue
authors.forename: Gerard J
authors.initials: GJ
authors.lastname: Bunick
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Chazin
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D064026:Calbindins; D020013:Calcium Signaling / Q000502:physiology*; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics; D018360:Crystallography, X-Ray; D015195:Drug Design; D020832:EF Hand Motifs / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D064030:S100 Calcium Binding Protein G / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D064026:Calbindins; D002147:Calmodulin; D064030:S100 Calcium Binding Protein G