Centrin is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. It is found in microtubule-organizing centers of organisms ranging from algae and yeast to man. In vitro, the C-terminal domain of centrin binds to the yeast centrosomal protein Kar1p in a calcium-dependent manner, whereas the N-terminal domain does not show any appreciable affinity for Kar1p. To obtain deeper insights into the structural basis for centrin's function, we have characterized the affinities of the C-terminal domain of Chlamydomonas reinhardtii centrin for calcium and for a peptide fragment of Kar1p using CD, fluorescence, and NMR spectroscopy. Calcium binding site IV in C. reinhardtii centrin was found to bind Ca2+ approximately 100-fold more strongly than site III. In the absence of Ca2+, the protein occupies a mixture of closed conformations. Binding of a single ion in site IV is sufficient to radically alter the conformational equilibrium, promoting occupancy of an open conformation. However, an exchange between closed and open conformations remains even at saturating levels of Ca2+. The population of the open conformation is substantially stabilized by the presence of the target peptide Kar1p-(239-257) to a point where a single ion bound in site IV is sufficient to completely shift the conformational equilibrium to the open conformation. This is reflected in the enhancement of the Ca2+ affinity in this site by more than an order of magnitude. These data confirm the direct coupling of the Ca2+ binding-induced shift in the equilibrium between the closed and open conformations to the binding of the peptide. Combined with the common localization of the two proteins in the microtubule organizing center, our results suggest that centrin is constitutively bound to Kar1p through its C-terminal domain and that centrin's calcium sensor activities are mediated by the N-terminal domain.

• PMID: 15452116
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Journal Article | Research Support, N.I.H., Extramural | Research Support, U.S. Gov't, P.H.S.

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Hu H, Sheehan JH, Chazin WJ

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