Martinez-Sanz Juan, Yang Ao, Blouquit Yves, Duchambon Patricia, Assairi Liliane, Craescu Constantin T
The Integrative Imaging Unit, INSERM U759/Institut Curie-Recherche, Centre Universitaire Paris-Sud, Orsay Cedex, France.
FEBS J. 2006 Oct;273(19):4504-15. doi: 10.1111/j.1742-4658.2006.05456.x. Epub 2006 Sep 5.
hSfi1, a human centrosomal protein with homologs in other eukaryotic organisms, includes 23 repeats, each of 23 amino acids, separated by 10 residue linkers. The main molecular partner in the centrosome is a small, calcium-binding EF-hand protein, the human centrin 2. Using isothermal titration calorimetry experiments, we characterized the centrin-binding capacity of three isolated hSfi1 repeats, two exhibiting the general consensus motif and the third being the unique Pro-containing human repeat. The two standard peptides bind human centrin 2 and its isolated C-terminal domain with high affinity (approximately 10(7) M(-1)) by an enthalpy-driven mechanism, with a moderate Ca2+ dependence. The Pro-containing repeat shows a binding affinity that is two orders of magnitude lower. The target binding site is localized within the C-terminal domain of human centrin 2. Fluorescence titration and NMR spectroscopy show that the well-conserved Trp residue situated in the C-terminus of each repeat is deeply embedded in a protein hydrophobic cavity, indicating that the peptide direction is reversed relative to previously studied centrin targets. The present results suggest that almost all of the repeats of the Sfi1 protein may independently bind centrin molecules. On the basis of this hypothesis and previous studies on centrin self-assembly, we propose a working model for the role of centrin-Sfi1 interactions in the dynamic structure of centrosome-associated contractile fibers.
hSfi1是一种在其他真核生物中具有同源物的人类中心体蛋白，包含23个重复序列，每个重复序列由23个氨基酸组成，中间间隔10个残基的连接子。中心体中的主要分子伴侣是一种小的、结合钙的EF手蛋白——人类中心蛋白2。通过等温滴定量热法实验，我们对三个分离的hSfi1重复序列与中心蛋白的结合能力进行了表征，其中两个呈现出一般的共有基序，第三个是独特的含脯氨酸的人类重复序列。这两个标准肽通过焓驱动机制以高亲和力（约10⁷ M⁻¹）结合人类中心蛋白2及其分离的C末端结构域，对Ca²⁺有适度依赖性。含脯氨酸的重复序列显示出低两个数量级的结合亲和力。靶标结合位点位于人类中心蛋白2的C末端结构域内。荧光滴定和核磁共振光谱表明，每个重复序列C末端保守的色氨酸残基深深嵌入蛋白质疏水腔中，这表明肽的方向相对于先前研究的中心蛋白靶标是相反的。目前的结果表明，Sfi1蛋白的几乎所有重复序列可能独立结合中心蛋白分子。基于这一假设以及先前对中心蛋白自组装的研究，我们提出了一个关于中心蛋白 - Sfi1相互作用在中心体相关收缩纤维动态结构中作用的工作模型。
