The elusive folding mechanism of KH1 domain of Fragile X messenger ribonucleoprotein 1

: K-homology (KH) domains are widespread RNA-binding modules characterized by a conserved fold despite low sequence identity. Among these, the KH domains of the Fragile X messenger ribonucleoprotein 1 (FMRP) play a key role in regulating mRNA translation in neurons, and their dysfunction, caused by missense mutations, is associated with Fragile X syndrome (FXS). In this work, by combining equilibrium and kinetic experiments, we investigate the folding mechanism of the KH1 domain of FMRP. Building on previous findings showing that the FXS-associated G266E mutation leads to complete unfolding of the domain, whereas the G266A substitution preserves the folding at the expense of stability, we performed the characterization of the folding mechanism of KH1 and the G266AKH1 variant over a range of pH conditions. Our results indicate that, although KH1 appears to follow a two-state folding mechanism, destabilizing perturbations, either induced by pH changes or by a single-site mutation, reveal the presence of a more complex folding landscape involving a high-energy intermediate. These findings highlight the critical role of residue 266 in maintaining KH1 structural stability while minimizing steric constraints and provide new insights into the folding landscape of KH domains.