Abstract：
The Fragile X messenger ribonucleoprotein (FMRP) is a multi-domain protein involved in interactions with various macromolecules, including proteins and coding/non-coding RNAs. The three KH domains (KH0, KH1 and KH2) within FMRP are recognized for their roles in mRNA binding. In the context of Fragile X syndrome (FXS), over-and-above CGG triplet repeats expansion, three specific point mutations have been identified, each affecting one of the three KH domains (R138QKH0, G266EKH1, and I304NKH2) resulting in the expression of non-functional FMRP. This study aims to elucidate the molecular mechanism underlying the loss of function associated with the G266EKH1 pathological variant. We investigate the conformational and dynamic properties of the isolated KH1 domain and the two KH1 site-directed mutants G266EKH1 and G266AKH1. Employing a combined in vitro and in silico approach, we reveal that the G266EKH1 variant lacks the characteristic features of a folded domain. This observation provides an explanation for functional impairment observed in FMRP carrying the G266E mutation within the KH1 domain, as it renders the domain unable to fold properly. Molecular Dynamics simulations suggest a pivotal role for residue 266 in regulating the structural stability of the KH domains, primarily through stabilizing the α-helices of the domain. Overall, these findings enhance our comprehension of the molecular basis for the dysfunction associated with the G266EKH1 variant in FMRP.
摘要：
脆性X信使核糖核蛋白（FMRP）是一个复杂的，参与与各种大分子相互作用的多结构域蛋白质，包括蛋白质和编码/非编码RNA。FMRP内的三个KH结构域(KH0、KH1和KH2)因其在mRNA结合中的作用而被识别。在脆性X综合征(FXS)的背景下，以上CGG三联体重复扩增，已经确定了三个特定的点突变，每个影响三个KH结构域（R138QKH0，G266EKH1和I304NKH2）之一，导致非功能性FMRP的表达。本研究旨在阐明与G266EKH1病理变体相关的功能丧失的分子机制。我们研究了分离的KH1结构域和两个KH1定点突变体G266EKH1和G266AKH1的构象和动力学特性。采用体外和计算机模拟相结合的方法，我们发现G266EKH1变体缺乏折叠域的特征性特征。这一观察结果为在KH1结构域内携带G266E突变的FMRP中观察到的功能损害提供了解释。因为它使域无法正确折叠。分子动力学模拟表明，残基266在调节KH结构域的结构稳定性方面具有关键作用，主要通过稳定结构域的α-螺旋。总的来说,这些发现增强了我们对FMRP中与G266EKH1变体相关的功能障碍的分子基础的理解.
