School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi, India.
PLoS One. 2023 Jan 19;18(1):e0278396. doi: 10.1371/journal.pone.0278396. eCollection 2023.
Human Replication Protein A (hRPA) is a multidomain protein that interacts with ssDNA intermediates to provide the latter much-needed stability during DNA metabolism and maintain genomic integrity. Although the ssDNA organization with hRPA was studied recently through experimental means, characterizing the underlying mechanism at the atomic level remains challenging because of the dynamic domain architecture of hRPA and poorly understood heterogeneity of ssDNA-protein interactions. Here, we used a computational framework, precisely tailored to capture protein-ssDNA interactions, and investigated the binding of hRPA with a 60 nt ssDNA. Two distinct binding mechanisms are realized based on the hRPA domain flexibility. For a rigid domain architecture of hRPA, ssDNA binds sequentially with hRPA domains, resulting in slow association kinetics. The binding pathway involves the formation of stable and distinct intermediate states. On contrary, for a flexible domain architecture of hRPA, ssDNA binds synergistically to the A and B domains followed by the rest of hRPA. The domain dynamics in hRPA alleviates the free energy cost of domain orientation necessary for specific binding with ssDNA, leading to fast association kinetics along a downhill binding free energy landscape. An ensemble of free energetically degenerate intermediate states is encountered that makes it arduous to characterize them structurally. An excellent match between our results with the available experimental observations provides new insights into the rich dynamics of hRPA binding to ssDNA and in general paves the way to investigate intricate details of ssDNA-protein interactions, crucial for cellular functioning.
人源复制蛋白 A（hRPA）是一种多功能蛋白，可与单链 DNA 中间体相互作用，在 DNA 代谢过程中为后者提供急需的稳定性，并维持基因组完整性。尽管最近通过实验手段研究了 hRPA 与 ssDNA 的相互作用结构，但由于 hRPA 动态的结构域架构以及对 ssDNA-蛋白相互作用的异质性理解不足，在原子水平上对其潜在机制进行描述仍然具有挑战性。在这里，我们使用了一个计算框架，该框架经过精确设计，可以捕获蛋白-ssDNA 相互作用，并研究了 hRPA 与 60 个核苷酸 ssDNA 的结合。基于 hRPA 结构域的灵活性，实现了两种不同的结合机制。对于刚性 hRPA 结构域架构，ssDNA 与 hRPA 结构域依次结合，导致缓慢的结合动力学。结合途径涉及到稳定和独特的中间状态的形成。相反，对于柔性 hRPA 结构域架构，ssDNA 协同结合 A 和 B 结构域，然后结合其余的 hRPA。hRPA 中的结构域动力学缓解了与 ssDNA 特异性结合所需的结构域定向的自由能成本，从而沿着下坡的结合自由能景观实现快速的结合动力学。遇到了一组在自由能上等价的中间状态，使得对它们的结构进行描述变得困难。我们的结果与现有实验观察结果之间的极好匹配，为 hRPA 与 ssDNA 结合的丰富动力学提供了新的见解，并为研究 ssDNA-蛋白相互作用的复杂细节铺平了道路，这些细节对细胞功能至关重要。
