Chen Ting, Yeh Hung-Wei, Chen Po-Pang, Huang Wei-Ting, Wu Chu-Ya, Liao Tzu-Chen, Lin Shiou-Lan, Chen Yi-Yun, Lin Kai-Ti, Hsu Shang-Te Danny, Cheng Hui-Chun
Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300044, Taiwan.
Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Biochim Biophys Acta Gen Subj. 2022 May;1866(5):130099. doi: 10.1016/j.bbagen.2022.130099. Epub 2022 Feb 5.
OLA1 is a P-loop ATPase, implicated in centrosome duplication through the interactions with tumor suppressors BRCA1 and BARD1. Disruption of the interaction of OLA1 with BARD1 results in centrosome amplification. However, the molecular interplay and mechanism of the OLA1-BARD1 complex remain elusive. Here, we use a battery of biophysical, biochemical, and structural analyses to elucidate the molecular basis of the OLA1-BARD1 interaction. Our structural and enzyme kinetics analyses show this nucleotide-dependent interaction enhances the ATPase activity of OLA1 by increasing the turnover number (k). Unlike canonical GTPase activating proteins that act directly on the catalytic G domain, the BARD1 BRCT domain binds to the OLA1 TGS domain via a highly conserved BUDR motif. A cancer related mutation V695L on BARD1 is known to associate with centrosome abnormality. The V695L mutation reduces the BARD1 BRCT-mediated activation of OLA1. Crystallographic snapshot of the BRCT V695L mutant at 1.88 Å reveals this mutation perturbs the OLA1 binding site, resulting in reduced interaction. Altogether, our findings suggest the BARD1 BRCT domain serves as an ATPase activating protein to control OLA1 allosterically.
OLA1是一种P环ATP酶，通过与肿瘤抑制因子BRCA1和BARD1相互作用参与中心体复制。OLA1与BARD1相互作用的破坏会导致中心体扩增。然而，OLA1 - BARD1复合物的分子相互作用和机制仍不清楚。在这里，我们使用一系列生物物理、生化和结构分析来阐明OLA1 - BARD1相互作用的分子基础。我们的结构和酶动力学分析表明，这种依赖核苷酸的相互作用通过增加周转数（k）来增强OLA1的ATP酶活性。与直接作用于催化G结构域的典型GTP酶激活蛋白不同，BARD1的BRCT结构域通过高度保守的BUDR基序与OLA1的TGS结构域结合。已知BARD1上的一个癌症相关突变V695L与中心体异常有关。V695L突变降低了BARD1的BRCT结构域介导的OLA1激活。BRCT V695L突变体在1.88 Å分辨率下的晶体学快照显示，该突变扰乱了OLA1结合位点，导致相互作用减弱。总之，我们的研究结果表明，BARD1的BRCT结构域作为一种ATP酶激活蛋白，通过变构作用控制OLA1。
