Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institut, 5232 Villigen, Switzerland.
Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), 1015 Lausanne, Switzerland.
Structure. 2016 Aug 2;24(8):1358-1371. doi: 10.1016/j.str.2016.06.011. Epub 2016 Jul 28.
Centrioles are microtubule-based structures that play important roles notably in cell division and cilium biogenesis. CEP135/Bld10p family members are evolutionarily conserved microtubule-binding proteins important for centriole formation. Here, we analyzed in detail the microtubule-binding activity of human CEP135 (HsCEP135). X-ray crystallography and small-angle X-ray scattering in combination with molecular modeling revealed that the 158 N-terminal residues of HsCEP135 (HsCEP135-N) form a parallel two-stranded coiled-coil structure. Biochemical, cryo-electron, and fluorescence microscopy analyses revealed that in vitro HsCEP135-N interacts with tubulin, protofilaments, and microtubules and induces the formation of microtubule bundles. We further identified a 13 amino acid segment spanning residues 96-108, which represents a major microtubule-binding site in HsCEP135-N. Within this segment, we identified a cluster of three lysine residues that contribute to the microtubule bundling activity of HsCEP135-N. Our results provide the first structural information on CEP135/Bld10p proteins and offer insights into their microtubule-binding mechanism.
中心体是一种基于微管的结构，在细胞分裂和纤毛发生中起着重要作用。CEP135/Bld10p 家族成员是进化上保守的微管结合蛋白，对中心体的形成很重要。在这里，我们详细分析了人类 CEP135（HsCEP135）的微管结合活性。X 射线晶体学和小角度 X 射线散射结合分子建模表明，HsCEP135 的 158 个 N 端残基（HsCEP135-N）形成平行的双股螺旋卷曲结构。生化、冷冻电镜和荧光显微镜分析表明，HsCEP135-N 在体外与微管蛋白、原纤维和微管相互作用，并诱导微管束的形成。我们进一步鉴定了一个跨越残基 96-108 的 13 个氨基酸片段，它代表了 HsCEP135-N 中的主要微管结合位点。在这个片段中，我们鉴定出三个赖氨酸残基簇，它们有助于 HsCEP135-N 的微管束形成活性。我们的结果提供了 CEP135/Bld10p 蛋白的第一个结构信息，并为它们的微管结合机制提供了见解。
