Cavities in Choline Acetyltransferase and Neuromuscular Disorders

Our goal is to understand the effects of congenital mutations in choline acetyltransferase (ChAT), which synthesizes the prototypical neurotransmitter acetylcholine. Decreases in ChAT activity are associated with a number of neurological disease states, and mutations in the enzyme cause a severe neuromuscular disorder known as congenital myasthenic syndrome with episodic apnea (CMS. Our crystal structure of ChAT showed that the known congenital mutations are distributed widely over the enzyme despite their common effects on function, and recent structures show that two of the mutations affect the conformation of the catalytic histidine residue. Further, the core residue packing in ChAT is poor, leaving an unusually large number of cavities (voids). Based on these observations, we hypothesize that the large number of cavities in ChAT makes it conformationally unstable, so that point mutations cause structural changes that propagate to disrupt function. We will use a combination of structural, biophysical, and cell biological approaches to test this hypothesis focusing on a cavity near the two congenital mutations that have been characterized structurally. Two specific aims are proposed: 1) characterize the role of an internal cavity in mediating the functional effects of two congenital mutations, 2) assess how filling the cavity effects structural changes associated with the congenital mutations This work will serve as an initial test of our hypothesis regarding the role of packing defects in ChAT, directly addressing the molecular mechanism underlying a human disease and guiding future studies of ChAT and the motor disorders associated with decreases in its function.