Humans have two class VII myosins, myosin VIIa and VIIb, which are very similar in domain structure except that myosin VIIb possesses a unique 19 amino acid insert at the actin-binding site and lacks a putative coiled-coil domain. Mutations in myosin VIIa have been linked to type 1b Usher syndrome, the most common deafness-blindness disorder in humans, and two other hearing loss disorders in humans, DFNB2 and DFNA11. Myosin VIIb is believed to traffic and organize membranes and membrane-associated proteins. Despite the critical roles class VII myosins play in cells, understanding the basis of myosin VII function and the deafness phenotype associated with myosin VIIa has been limited by a lack of information regarding the chemical and physical properties of myosin VII. Efforts in this proposal focus on understanding the enzymatic and motor properties of myosin VIIa and VIIb.
Aim 1 will determine if full-length myosin VIIa and VIIb are monomers or dimers.
Aim 2 will provide a complete kinetic characterization of the myosin VIIa and VIIb ATPase cycles, determine the contributions of the 19 amino acid insertion to the ATPase cycle kinetics through the generation of mutants and chimeric proteins, and identify how mutations that generate deafness affect myosin VIIa motor activity.
Aim 3 will measure how external loads modulate ADP and actin binding to high duty ratio myosins and evaluate mechanisms of strain-dependent head-head coordination.
Aim 4 will employ high-resolution spectroscopic methods to measure cooperative changes in the structural dynamics of actin filaments induced by high duty ratio myosin binding. The two highly conserved isoforms provide a unique opportunity to test the structural and functional effects of the actin-binding loop and of dimerization on myosin function. The proposed detailed analysis of reaction mechanisms and structural dynamics will help develop testable hypotheses regarding the function and mechanism of myosin VII, and illustrate how sequence and structural variations in myosin motors define their enzymatic properties, which ultimately dictates their biological functions. Because mutations and disruption of myosin VIIa function are of clinical relevance, the proposed experiments will advance our understanding of the molecular basis of several human diseases.
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