Abstract

The structure and mechanism of action of a family of myristoylated calcium-sensing proteins, exemplified by retinal recoverin, will be investigated by fluorescence, NMR, and spin-label spectroscopy. These acylated proteins move from the cytosol to a membrane on binding calcium.
The aim i s to determine the molecular mechanism of calcium-myristoyl switches and define their roles in signal transduction processes. Having solved the three-dimensional structures of calcium-free myristoylated recoverin and calcium-bound unmyristoylated recoverin, we turn now to the determination of the structure of the calcium-bound form of myristoylated recoverin to reveal the precise structural basis of the switch. Our working hypothesis is that recoverin and brain homologs such as neurocalcin and hippocalcin serve to couple calcium cascades to G-protein cascades by interacting with kinases that deactivate seven-helix receptors. The membrane-contact sites of recoverin in the Ca2+-bound state and their depth will be determined by spin-label ESR spectroscopy using nitroxide-labeled myristate and nitroxide-tagged cysteines introduced by mutagenesis. Structural, spectroscopic, and functional studies of the interaction of Ca2+-bound recoverin with rhodopsin kinase and fragments of this target will also be carried out. Three classes of recoverin mutants will be generated and analyzed to further our understanding of the structure and dynamics of this sensor: (1) Nonpolar residues in the myristoyl binding pocket will be replaced by polar ones. (2) Glycines in putative hinge regions will be changed to alanine. (3) Charged residues surrounding a concave hydrophobic surface, the putative target-binding site, will be charged to oppositely-charged residues. Structural studies of neurocalcin and hippocalcin will also be carried out and targets of these brain homologs will be identified using the yeast two-hybrid system. A yeast homolog will be expressed and analyzed to provide insight into the evolution of this family of neuronal calcium sensors.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024032-21
Application #
2684685
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1979-01-01
Project End
1999-06-30
Budget Start
1998-04-01
Budget End
1999-06-30
Support Year
21
Fiscal Year
1998
Total Cost
Indirect Cost

  Institution

Name
Stanford University
Department
Biology
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305

  Publications

Tanaka, T; Ames, J B; Kainosho, M et al. (1998) Differential isotype labeling strategy for determining the structure of myristoylated recoverin by NMR spectroscopy. J Biomol NMR 11:135-52
Baldwin, A N; Ames, J B (1998) Core mutations that promote the calcium-induced allosteric transition of bovine recoverin. Biochemistry 37:17408-19
Ames, J B; Tanaka, T; Stryer, L et al. (1996) Portrait of a myristoyl switch protein. Curr Opin Struct Biol 6:432-8
Boniface, J J; Lyons, D S; Wettstein, D A et al. (1996) Evidence for a conformational change in a class II major histocompatibility complex molecule occurring in the same pH range where antigen binding is enhanced. J Exp Med 183:119-26
Ladant, D (1995) Calcium and membrane binding properties of bovine neurocalcin delta expressed in Escherichia coli. J Biol Chem 270:3179-85
Shopes, B (1995) Temperature-dependent binding of IgG1 to a human high affinity Fc receptor. Mol Immunol 32:375-8
Zozulya, S; Ladant, D; Stryer, L (1995) Expression and characterization of calcium-myristoyl switch proteins. Methods Enzymol 250:383-93
Ames, J B; Porumb, T; Tanaka, T et al. (1995) Amino-terminal myristoylation induces cooperative calcium binding to recoverin. J Biol Chem 270:4526-33
Ames, J B; Tanaka, T; Ikura, M et al. (1995) Nuclear magnetic resonance evidence for Ca(2+)-induced extrusion of the myristoyl group of recoverin. J Biol Chem 270:30909-13
Hanson, P I; Meyer, T; Stryer, L et al. (1994) Dual role of calmodulin in autophosphorylation of multifunctional CaM kinase may underlie decoding of calcium signals. Neuron 12:943-56

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