Mondal Balaka, Nagesh Jayashree, Reddy Govardhan
Solid State and Structural Chemistry Unit, Indian Institute of Science, Bengaluru, Karnataka, India 560012.
J Phys Chem B. 2021 Feb 25;125(7):1705-1715. doi: 10.1021/acs.jpcb.0c07833. Epub 2021 Feb 10.
Human γD (HγD) and γC (HγC) are two-domain crystallin (Crys) proteins expressed in the nucleus of the eye lens. Structural perturbations in the protein often trigger aggregation, which eventually leads to cataract. To decipher the underlying molecular mechanism, it is important to characterize the partially unfolded conformations, which are aggregation-prone. Using a coarse grained protein model and molecular dynamics simulations, we studied the role of on-pathway folding intermediates in the early stages of aggregation. The multidimensional free energy surface revealed at least three different folding pathways with the population of partially structured intermediates. The two dominant pathways confirm sequential folding of the N-terminal [Ntd] and the C-terminal domains [Ctd], while the third, least favored, pathway involves intermediates where both the domains are partially folded. A native-like intermediate (*), featuring the folded domains and disrupted interdomain contacts, gets populated in all three pathways. * forms domain swapped dimers by swapping the entire Ntds and Ctds with other monomers. Population of such oligomers can explain the increased resistance to unfolding resulting in hysteresis observed in the folding experiments of HγD Crys. An ensemble of double domain swapped dimers are also formed during refolding, where intermediates consisting of partially folded Ntds and Ctds swap secondary structures with other monomers. The double domain swapping model presented in our study provides structural insights into the early events of aggregation in Crys proteins and identifies the key secondary structural swapping elements, where introducing mutations will aid in regulating the overall aggregation propensity.
人类γD（HγD）和γC（HγC）是在晶状体细胞核中表达的双结构域晶状体蛋白（Crys）。蛋白质中的结构扰动常常引发聚集，最终导致白内障。为了解析潜在的分子机制，表征易于聚集的部分未折叠构象非常重要。我们使用粗粒度蛋白质模型和分子动力学模拟，研究了聚集早期阶段的折叠中间态在其中的作用。多维自由能表面揭示了至少三种不同的折叠途径以及部分结构化中间态的丰度。两条主要途径证实了N端结构域（Ntd）和C端结构域（Ctd）的顺序折叠，而第三条最不常见的途径涉及两个结构域都部分折叠的中间态。一种具有折叠结构域和破坏的结构域间接触的类天然中间态（*）在所有三条途径中都有出现。*通过与其他单体交换整个Ntd和Ctd形成结构域交换二聚体。这种寡聚体的丰度可以解释在HγD Crys折叠实验中观察到的对去折叠的抗性增加以及滞后现象。在重折叠过程中还形成了一组双结构域交换二聚体，其中由部分折叠的Ntd和Ctd组成的中间态与其他单体交换二级结构。我们研究中提出的双结构域交换模型为Crys蛋白聚集的早期事件提供了结构见解，并确定了关键的二级结构交换元件，在这些元件处引入突变将有助于调节整体聚集倾向。
