Allele-specific conformational rescue of KIF1A(T99M) by genetic suppressors in a C. elegans model of KIF1A-associated neurological disorder.

KIF1A-associated neurological disorder (KAND) arises from mutations in the microtubule motor KIF1A, disrupting synaptic vesicle transport. Here, we investigate the pathogenic T99M substitution in KIF1A's P-loop, which induces steric hindrance, impairing ATP/ADP coordination and motor activity. Using CRISPR-engineered C. elegans expressing the homologous UNC-104(T95M) mutation, we conducted forward genetic screens and identified recurrent intragenic suppressors (T95V/I) that restored animal motility and synaptic vesicle distribution. Molecular dynamics simulations revealed that replacing methionine with valine/isoleucine alleviated steric clashes in the nucleotide-binding pocket and stabilized Mg²⁺-ATP coordination. Biochemical assays showed that T95V/I partially recovered microtubule gliding velocity and processivity, demonstrating that even modest motor reactivation mitigates neuronal dysfunction. Inspired by prior success with fisetin in rescuing the KIF1A R11Q variant, we propose allele-specific conformational stabilization as a therapeutic strategy for KAND. Our findings highlight the structural plasticity of motor domain and provide a framework for precision therapies targeting pathogenic variants through genetic suppressors.