Structural independence of the two EF-hand domains of caltractin.

Veeraraghavan S, Fagan PA, Hu H, Lee V, Harper JF, Huang B, Chazin WJ
J Biol Chem. 2002 277 (32): 28564-71

PMID: 12034713 · DOI:10.1074/jbc.M112232200

Caltractin (centrin) is a member of the calmodulin subfamily of EF-hand Ca2+-binding proteins that is an essential component of microtubule-organizing centers in many organisms ranging from yeast and algae to humans. The protein contains two homologous EF-hand Ca2+-binding domains linked by a flexible tether; each domain is capable of binding two Ca2+ ions. In an effort to search for domain-specific functional properties of caltractin, the two isolated domains were subcloned and expressed in Escherichia coli. Ca2+ binding affinities and the Ca2+ dependence of biophysical properties of the isolated domains were monitored by UV, CD, and NMR spectroscopy. Comparisons to the corresponding results for the intact protein showed that the two domains function independently of each other in these assays. Titration of a peptide fragment from the yeast Kar1p protein to the isolated domains and intact caltractin shows that the two domains interact in a Ca2+-dependent manner, with the C-terminal domain binding much more strongly than the N-terminal domain. Measurements of the macroscopic Ca2+ binding constants show that only the N-terminal domain has sufficient apparent Ca2+ affinity in vitro (1-10 microm) to be classified as a traditional calcium sensor in signal transduction pathways. However, investigation of the microscopic Ca2+ binding events in the C-terminal domain by NMR spectroscopy revealed that the observed macroscopic binding constant likely results from binding to two sites with very different affinities, one in the micromolar range and the other in the millimolar range. Thus, the C-terminal domain appears to also be capable of sensing Ca2+ signals but is activated by the binding of a single ion.

MeSH Terms (25)

Amino Acid Sequence Animals Calcium Calcium-Binding Proteins Chlamydomonas Chromosomal Proteins, Non-Histone Circular Dichroism Cloning, Molecular Dose-Response Relationship, Drug Escherichia coli Fungal Proteins Hydrogen-Ion Concentration Ions Kinetics Magnetic Resonance Spectroscopy Molecular Sequence Data Nuclear Proteins Protein Binding Protein Conformation Protein Structure, Tertiary Recombinant Proteins Saccharomyces cerevisiae Proteins Sequence Homology, Amino Acid Spectrometry, Fluorescence Ultraviolet Rays

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