An S752D activation loop mutation dynamically primes Muscle-Specific Kinase for activation.

Muscle-Specific Kinase (MuSK) is a receptor tyrosine kinase essential for neuromuscular junction (NMJ) formation and maintenance, yet its regulation remains poorly understood. Crystallographic studies of wild-type MuSK revealed an autoinhibited conformation with tyrosines in the activation loop (A-loop) anchored within the catalytic cleft to stabilize the closed, inactive conformation. We showed previously that additional phosphorylation of an A-loop serine may 'prime' MuSK for activation to sensitize it to ligand(s) in certain settings. Here, we employed crystallography, biochemical assays, and hydrogen-deuterium exchange and mass spectrometry (HDX-MS) to test this hypothesis. We found that introducing a phosphomimetic S752D mutation disrupts autoinhibitory A-loop interactions to increase ATP-binding affinity and catalytic turnover. Using HDX-MS, we further observed that the S752D mutation increases A-loop structural flexibility to relieve autoinhibition. The S752D mutation also stabilizes the juxtamembrane NPXY motif region, a docking site for the adaptor Dok7, possibly priming MuSK for downstream signaling. Together, these findings reveal dynamic transitions that underlie relief of MuSK autoinhibition and provide a mechanistic framework for understanding MuSK activation at the NMJ.