Impact of Neurodevelopmental Disorder-Associated Mutations on DDX3X Structure and Oligomerization

Introduction: DDX3X is a member of the DEAD-box helicase family, primarily implicated in neurodevelopment and neurodevelopmental disorders (NDD). This protein contains a structurally conserved helicase core formed by two RecA-like domains (D1 and D2) connected via a flexible linker. These motifs mediate key functions such as ATP hydrolysis and RNA unwinding. Intrinsically disordered regions (IDRs) flank the helicase core at both the N- and C-terminal ends. These IDRs also mediate DDX3X phase separation. Mutations in the helicase domain (R326H and R376C) have been linked to NDDs.

Objectives: The aim of this study is to investigate how NDD-associated mutations impact the structure and function of DDX3X.

Methods: We employed an integrative approach that combines NMR, X-ray crystallography, ATPase and RNA helicase activity assays, fluorescence microscopy, and ThT fluorescence spectroscopy. 

Results and Discussion: We demonstrated that both R326H in the D1 domain and R376C in the D2 domain locally disturb the protein structure, consequently altering enzymatic functions. We further observed that, while the R326H mutation results in the formation of smaller, liquid-like homotypic condensates in-vitro as compared to the wild-type protein, the R376C mutation leads to progressive condensate aggregation over time. Moreover, we observed that the R376C mutant tends to form higher order oligomers as compared to dimers observed for the wild-type full-length protein in the presence and absence of dsRNA. ThT spectroscopy indicated that the R376H mutation in the full-length protein leads to beta-amyloid structure formation.

Conclusion: Together, our results provide new insights into the molecular mechanisms of DDX3X condensation and aggregation.