DNA Replication and Repair

在我们之前的研究中，我们已经解决了FAM111A丝氨酸蛋白酶结构域（SPD）的x射线晶体结构。我们的生化和结构研究表明FAM111A SPD通过n端α -1螺旋形成同型二聚体，其中两个亚基通过疏水相互作用结合在一起。在体外实验中，用天冬氨酸取代二聚界面上的缬氨酸残基减少了SPD二聚体的形成和蛋白酶活性，以及在细胞中保护复制叉免受拓扑异构酶抑制剂喜树碱诱导的DPCs的能力。为了了解SPD的二聚化是如何促进蛋白酶活性的，我们解决了SPD突变体缺乏二聚化界面的x射线晶体结构。有趣的是，在单体突变体中，不仅二聚化界面，而且对蛋白酶活性至关重要的活性位点结构氧阴离子孔也被扰乱。这些新数据揭示了FAM111A SPD二聚化依赖性活性的机制，其中二聚化引发了一系列无序到有序的转变，从而导致氧阴离子空穴的稳定。这些结构研究将帮助我们确定调节FAM111A在DPCs中停滞的复制叉上的蛋白酶活性的潜在机制，并促进FAM111A抑制剂作为抗肿瘤治疗药物（包括拓扑异构酶抑制剂）的增敏剂的开发。

In our previous study, we have solved X-ray crystal structures of the FAM111A serine protease domain (SPD). Our biochemical and structural studies suggested that FAM111A SPD forms a homodimer through the alpha-1 helix at the N-terminus, where two subunits are held together through hydrophobic interactions. Substitution of the valine residue on the dimerization interface with aspartate diminished dimer formation and protease activity of the SPD in vitro, as well as the ability to protect replication forks from DPCs induced by a topoisomerase inhibitor, camptothecin, in cells. To understand how dimerization of the SPD promotes the protease activity, we have solved X-ray crystal structures of an SPD mutant that lacks a part of the dimerization interface. Interestingly, not only the dimerization interface, but also the oxyanion hole, an active site structure critical for protease activity, is disordered in the monomeric mutants. These new data reveal a mechanism of the dimerization-dependent activity of the FAM111A SPD, in which dimerization triggers a cascade of disorder-to-order transitions that leads to the stabilization of the oxyanion hole. These structural studies will help us determine potential mechanisms that regulate the protease activity of FAM111A at replication forks stalled at DPCs and facilitate the development of FAM111A inhibitors as a sensitizers of anti-cancer therapeutics including topoisomerase inhibitors.