From the Departments of Biology and.
Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599.
J Biol Chem. 2014 Jul 25;289(30):20727-39. doi: 10.1074/jbc.M114.576041. Epub 2014 Jun 11.
Centrioles play a key role in nucleating polarized microtubule networks. In actively dividing cells, centrioles establish the bipolar mitotic spindle and are essential for genomic stability. Drosophila anastral spindle-2 (Ana2) is a conserved centriole duplication factor. Although recent work has demonstrated that an Ana2-dynein light chain (LC8) centriolar complex is critical for proper spindle positioning in neuroblasts, how Ana2 and LC8 interact is yet to be established. Here we examine the Ana2-LC8 interaction and map two LC8-binding sites within the central region of Ana2, Ana2M (residues 156-251). Ana2 LC8-binding site 1 contains a signature TQT motif and robustly binds LC8 (KD of 1.1 μm), whereas site 2 contains a TQC motif and binds LC8 with lower affinity (KD of 13 μm). Both LC8-binding sites flank a predicted ~34-residue α-helix. We present two independent atomic structures of LC8 dimers in complex with Ana2 LC8-binding site 1 and site 2 peptides. The Ana2 peptides form β-strands that extend a central composite LC8 β-sandwich. LC8 recognizes the signature TQT motif in the first LC8 binding site of Ana2, forming extensive van der Waals contacts and hydrogen bonding with the peptide, whereas the Ana2 site 2 TQC motif forms a uniquely extended β-strand, not observed in other dynein light chain-target complexes. Size exclusion chromatography coupled with multiangle static light scattering demonstrates that LC8 dimers bind Ana2M sites and induce Ana2 tetramerization, yielding an Ana2M4-LC88 complex. LC8-mediated Ana2 oligomerization probably enhances Ana2 avidity for centriole-binding factors and may bridge multiple factors as required during spindle positioning and centriole biogenesis.
中心体在形成极化微管网络中起着关键作用。在活跃分裂的细胞中，中心体建立双极有丝分裂纺锤体，对于基因组稳定性至关重要。果蝇无星纺锤体-2（Ana2）是一种保守的中心体复制因子。尽管最近的工作表明，Ana2-动力蛋白轻链（LC8）中心体复合物对于神经母细胞中正确的纺锤体定位至关重要，但 Ana2 和 LC8 如何相互作用尚未确定。在这里，我们研究了 Ana2-LC8 的相互作用，并在 Ana2 的中心区域内定位了两个 LC8 结合位点，即 Ana2M（残基 156-251）。Ana2 LC8 结合位点 1 包含一个特征性的 TQT 基序，并且可以强烈结合 LC8（KD 值为 1.1μm），而位点 2 包含一个 TQC 基序，与 LC8 的结合亲和力较低（KD 值为 13μm）。这两个 LC8 结合位点都位于一个预测的~34 个残基的α-螺旋侧翼。我们展示了 LC8 二聚体与 Ana2 LC8 结合位点 1 和位点 2 肽复合物的两个独立原子结构。Ana2 肽形成延伸中央复合 LC8 β-三明治的β-链。LC8 识别 Ana2 第一个 LC8 结合位点中的特征性 TQT 基序，与肽形成广泛的范德华接触和氢键，而 Ana2 位点 2 的 TQC 基序形成独特延伸的β-链，在其他动力蛋白轻链-靶复合物中未观察到。尺寸排阻色谱法结合多角度静态光散射表明，LC8 二聚体结合 Ana2M 位点并诱导 Ana2 四聚化，生成 Ana2M4-LC88 复合物。LC8 介导的 Ana2 寡聚化可能增强了 Ana2 对中心体结合因子的亲和力，并可能在纺锤体定位和中心体发生过程中作为桥梁连接多个因子。
