Markov State Models and Molecular Dynamics Simulations Reveal the Conformational Transition of the Intrinsically Disordered Hypervariable Region of K‑Ras4B to the Ordered Conformation

K-Ras4B, the most frequently mutated Ras isoform in human tumors, plays a vital part in cell growth, differentiation, and survival. Its tail, the C-terminal hypervariable region (HVR), is involved in anchoring K-Ras4B at the cellular plasma membrane and in isoform-specific protein–protein interactions and signaling. In the inactive guanosine diphosphate-bound state, the intrinsically disordered HVR interacts with the catalytic domain at the effector-binding region, rendering K-Ras4B in its autoinhibited state. Activation releases the HVR from the catalytic domain, with its ensemble favoring an ordered α-helical structure. The large-scale conformational transition of the HVR from the intrinsically disordered to the ordered conformation remains poorly understood. Here, we deploy a computational scheme that integrates a transition path-generation algorithm, extensive molecular dynamics simulation, and Markov state model analysis to investigate the conformational landscape of the HVR transition pathway. Our findings reveal a stepwise pathway for the HVR transition and uncover several key conformational substates along the transition pathway. Importantly, key interactions between the HVR and the catalytic domain are unraveled, highlighting the pathogenesis of K-Ras4B mild mutations in several congenital developmental anomaly syndromes. Together, these findings provide a deeper understanding of the HVR transition mechanism and the regulation of K-Ras4B activity at an atomic level.K-Ras4B是人类肿瘤中突变最为频发的Ras亚型（Ras isoform），在细胞增殖、分化与存活过程中发挥关键作用。其C端尾部——C端高变区（C-terminal hypervariable region, HVR），负责将K-Ras4B锚定在细胞质膜上，并介导亚型特异性的蛋白质-蛋白质相互作用与信号转导。在失活的鸟苷二磷酸结合态下，内在无序的HVR会在效应物结合区与催化结构域发生相互作用，使K-Ras4B处于自抑制状态。激活过程会使HVR从催化结构域上解离，此时其构象系综更倾向于形成有序的α螺旋结构。HVR从内在无序到有序构象的大规模构象转变机制，目前仍未被充分阐明。本研究采用整合了过渡路径生成算法、大规模分子动力学模拟与马尔可夫状态模型（Markov state model）分析的计算策略，对HVR转变通路的构象景观展开研究。本研究揭示了HVR转变的分步通路，并鉴定出转变通路沿线的多个关键构象亚态。尤为重要的是，本研究阐明了HVR与催化结构域之间的关键相互作用，为解析多种先天性发育异常综合征中K-Ras4B轻度突变的致病机制提供了依据。综上，本研究成果从原子层面加深了我们对HVR转变机制以及K-Ras4B活性调控的理解。