Theory of Condensed Matter Group, Cavendish Laboratory, University of Cambridge, Cambridge, UK.
PLoS Comput Biol. 2011 Jul;7(7):e1002096. doi: 10.1371/journal.pcbi.1002096. Epub 2011 Jul 14.
The breast cancer suppressor BRCA2 controls the recombinase RAD51 in the reactions that mediate homologous DNA recombination, an essential cellular process required for the error-free repair of DNA double-stranded breaks. The primary mode of interaction between BRCA2 and RAD51 is through the BRC repeats, which are ∼35 residue peptide motifs that interact directly with RAD51 in vitro. Human BRCA2, like its mammalian orthologues, contains 8 BRC repeats whose sequence and spacing are evolutionarily conserved. Despite their sequence conservation, there is evidence that the different human BRC repeats have distinct capacities to bind RAD51. A previously published crystal structure reports the structural basis of the interaction between human BRC4 and the catalytic core domain of RAD51. However, no structural information is available regarding the binding of the remaining seven BRC repeats to RAD51, nor is it known why the BRC repeats show marked variation in binding affinity to RAD51 despite only subtle sequence variation. To address these issues, we have performed fluorescence polarisation assays to indirectly measure relative binding affinity, and applied computational simulations to interrogate the behaviour of the eight human BRC-RAD51 complexes, as well as a suite of BRC cancer-associated mutations. Our computational approaches encompass a range of techniques designed to link sequence variation with binding free energy. They include MM-PBSA and thermodynamic integration, which are based on classical force fields, and a recently developed approach to computing binding free energies from large-scale quantum mechanical first principles calculations with the linear-scaling density functional code onetep. Our findings not only reveal how sequence variation in the BRC repeats directly affects affinity with RAD51 and provide significant new insights into the control of RAD51 by human BRCA2, but also exemplify a palette of computational and experimental tools for the analysis of protein-protein interactions for chemical biology and molecular therapeutics.
乳腺癌抑制因子 BRCA2 控制着参与同源 DNA 重组反应的重组酶 RAD51，而同源 DNA 重组是细胞内必需的过程，对于 DNA 双链断裂的无差错修复至关重要。BRCA2 和 RAD51 之间的主要相互作用模式是通过 BRC 重复序列实现的，BRC 重复序列是约 35 个残基的肽基序，在体外可直接与 RAD51 相互作用。人类 BRCA2 与其哺乳动物同源物一样，包含 8 个 BRC 重复序列，其序列和间隔在进化上是保守的。尽管它们的序列保守，但有证据表明，不同的人类 BRC 重复序列具有不同的结合 RAD51 的能力。之前发表的晶体结构报告了人源 BRC4 与 RAD51 催化核心结构域之间相互作用的结构基础。然而，目前尚无关于其余七个 BRC 重复序列与 RAD51 结合的结构信息，也不知道为什么 BRC 重复序列在与 RAD51 结合的亲和力上表现出明显的差异，尽管它们的序列仅略有变化。为了解决这些问题，我们进行了荧光偏振测定，以间接测量相对结合亲和力，并应用计算模拟来研究八个人类 BRC-RAD51 复合物以及一组 BRC 癌症相关突变的行为。我们的计算方法涵盖了一系列旨在将序列变化与结合自由能联系起来的技术。它们包括基于经典力场的 MM-PBSA 和热力学积分，以及最近开发的一种从基于密度泛函理论的 onetep 线性标度量子力学第一性原理计算中计算结合自由能的方法。我们的研究结果不仅揭示了 BRC 重复序列中的序列变化如何直接影响与 RAD51 的亲和力，并为理解人类 BRCA2 对 RAD51 的控制提供了新的重要见解，而且还为化学生物学和分子治疗学中的蛋白质-蛋白质相互作用分析提供了一系列计算和实验工具。
