UBA5 missense variants disrupt UFM1 activation: Structural, dynamic, and functional dissection.

UFMylation is an evolutionarily conserved ubiquitin-like modification essential for cellular homeostasis. UBA5 acts as the sole E1 enzyme initiating this process, and biallelic UBA5 variants cause severe neurodevelopmental disorders. However, the specific mechanisms by which missense variants disrupt UFM1 activation remain unclear. Here, we applied AlphaFold2/3 modeling and molecular dynamics simulations to examine conformational states of UBA5 during activation, focusing on 11 clinically relevant missense variants within the adenylation domain. Structural analysis revealed that variants impair key mechanistic steps: monomer stabilization exemplified by p.Arg72Cys and p.Gly168Glu, dimerization disrupted by p.Val260Met, ATP coordination compromised in p.Arg55His, UFM1 recruitment weakened in p.Cys303Arg, and thioester bond formation impaired in p.Leu254Pro.Molecular dynamics simulations revealed variant-specific perturbations across activation stages: p.Arg72Cys and p.Gly168Glu increased flexibility and solvent exposure in the monomeric core; p.Val260Met and p.Leu254Pro altered dimer interface packing; p.Cys303Arg expanded and destabilized the UFM1-binding interface, while p.Arg55His reduced local flexibility near the ATP-binding pocket yet maintained global compactness. Biochemical and cellular assays, including thermal stability, ATP-binding, UFM1 charging, and substrate UFMylation, confirmed these defects and aligned with computational predictions. This study defines how individual UBA5 variants compromise structural transitions required for UFM1 activation and establishes a mechanistic framework linking variant-specific disruptions to disease pathogenesis.