Designing sequence to control protein function in an EF-hand protein.

Bunick CG, Nelson MR, Mangahas S, Hunter MJ, Sheehan JH, Mizoue LS, Bunick GJ, Chazin WJ
J Am Chem Soc. 2004 126 (19): 5990-8

PMID: 15137763 · DOI:10.1021/ja0397456

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.

MeSH Terms (14)

Amino Acids Amino Acid Sequence Calbindins Calcium Signaling Calmodulin Crystallography, X-Ray Drug Design EF Hand Motifs Models, Molecular Molecular Sequence Data Mutation Protein Conformation S100 Calcium Binding Protein G Structure-Activity Relationship

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