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

Area of Science:

• Biochemistry
• Structural Biology
• Genetics

Background:

• UFMylation is a vital ubiquitin-like modification for cellular homeostasis.
• UBA5 is the E1 enzyme initiating UFMylation; its variants cause severe neurodevelopmental disorders.
• Mechanisms of UBA5 missense variants impairing UFM1 activation are not fully understood.

Purpose of the Study:

• To investigate the structural and mechanistic effects of 11 clinically relevant UBA5 missense variants on UFM1 activation.
• To elucidate how these variants disrupt key steps in the UBA5 activation process.

Main Methods:

• Computational modeling using AlphaFold2/3 and molecular dynamics simulations to analyze UBA5 conformational states.
• Biochemical assays including thermal stability, ATP-binding, UFM1 charging, and substrate UFMylation.
• Cellular assays to validate computational predictions and assess functional consequences.

Main Results:

• Missense variants were found to impair UBA5 monomer stabilization, dimerization, ATP coordination, UFM1 recruitment, and thioester bond formation.
• Molecular dynamics revealed variant-specific impacts on flexibility, dimer interface, and UFM1-binding interface.
• Computational predictions were confirmed by biochemical and cellular assays, showing functional defects.

Conclusions:

• This study defines how specific UBA5 variants disrupt structural transitions essential for UFM1 activation.
• A mechanistic framework is established linking variant-specific structural defects to neurodevelopmental disease pathogenesis.