The Shaker-1 Mouse Myosin VIIa has a Drastically Compromised ATP Hydrolysis Mechanism

Abstract

The shaker-1 mice carry a missense mutation in the myosin VIIa gene (R502P) and exhibit deafness, circling behavior and mild retinal degeneration. Developmental and electrophysiological studies have shown that the cochlear function is altered in these mice. We carried out a detailed kinetic analysis of the baculoviral expressed truncated shaker-1 construct to assess whether the ATP hydrolysis mechanism is altered in these mice. The basal steady-state rate (0.03 s−1) is activated by actin only 1.5 -fold. ATP binding is relatively fast (1.22 uM−1s−1) and the dissociation of the myosin from the actomyosin complex is the same as for the wild type myosin VIIa (400 s−1). ADP binding to (1.8 uM−1s−1) and the dissociation (1.5 s−1) from the actomyosin complex are also similar to the wild type. Phosphate release from the M-ADP-Pi complex is slow (0.07 s−1) and is not activated by actin. Quenched flow experiments show that there is no Pi burst and the rate of the hydrolysis is 0.12 s−1. The R502 residue is in a small loop that is in the middle of an α-helical region which aligns with the switch-2 helix and is the continuation of the relay-loop. The sequence in the loop is changed from NRPM to NPPM which is likely to alter the conformation of the helix-loop-helix structure that is relatively straight in all myosins.Consequently the rate constants of the steps leading into the power stroke (hydrolysis and phosphate dissociation) are reduced greater than 10-fold whereas the post-power stoke steps are essentially unchanged. As a result the shaker-1 mutant protein has a severely compromised ATP hydrolysis mechanism in which < 10% of the normal fraction of the force-producing crossbridges are formed.Supported by NIH/RO3DC009335.