Conformational and dynamic properties of the KH1 domain of FMRP and its fragile X syndrome linked G266E variant

: 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.