The shaker-1 mouse myosin VIIa deafness mutation results in a severely reduced rate of the ATP hydrolysis step

Ailian Xiong,Jessica Haithcock,Yingying Liu,Lauren Eusner,Matthew Mcconnell,Howard D White,Betty Belknap,Eva Forgacs

doi:10.1074/jbc.m117.810119

Abstract

Mutations in the MYO7A gene, encoding the motor protein myosin VIIa, can cause Usher 1B, a deafness/blindness syndrome in humans, and the shaker-1 phenotype, characterized by deafness, head tossing, and circling behavior, in mice. Myosin VIIa is responsible for tension bearing and the transduction mechanism in the stereocilia and for melanosome transport in the retina, in line with the phenotypic outcomes observed in mice. However, the effect of the shaker-1 mutation, a R502P amino acid substitution, on the motor function is unclear. To explore this question, we determined the kinetic properties and the effect on the filopodial tip localization of the recombinant mouse myosin VIIa-5IQ-SAH R502P (myoVIIa-sh1) construct. Interestingly, although residue 502 is localized to a region thought to be involved in interacting with actin, the kinetic parameters for actin binding changed only slightly for the mutant construct. However, the rate constant for ATP hydrolysis (k+H + k-H) was reduced by ∼200-fold from 12 s-1 to 0.05 s-1, making the hydrolysis step the rate-limiting step of the ATPase cycle in the presence and absence of actin. Given that wild-type mouse myosin VIIa is a slow, high-duty ratio, monomeric motor, this altered hydrolysis rate would reduce activity to extremely low levels. Indeed, the translocation to the filopodial tips was hampered by the diminished motor function of a dimeric construct of the shaker-1 mutant. We conclude that the diminished motor activity of this mutant is most likely responsible for impaired hearing in the shaker-1 mice.

Highlights

Mutations in the MYO7A gene, encoding the motor protein myosin VIIa, can cause Usher 1B, a deafness/blindness syndrome in humans, and the shaker-1 phenotype, characterized by deafness, head tossing, and circling behavior, in mice
Myosin VIIa is responsible for tension bearing and the transduction mechanism in the stereocilia and for melanosome transport in the retina, in line with the phenotypic outcomes observed in mice
Myosin VIIa is an unconventional myosin that has a motor domain, a 5IQ neck domain, a single-␣-helix (SAH)2 domain, two large repeats of a myosin tail homology 4 (MyTH4) domain, an SH3 domain, and two 4.1-ezrin-radixin-moesin (FERM) domains that may function as a cargo-binding site [12, 13]

Summary

To whom correspondence should be addressed

The mutated proline residue for the shaker-1 myosin VIIa mutant is circled in red. Patients carrying MYO7A mutations suffer from Usher syndrome type 1B, which presents as hereditary deafness and progressive retinal degeneration (retinitis pigmentosa) [28], and two autosomal recessive disorders associated with non-syndromic hearing loss, DFNB2 and DFNA11 (29 –31). Mutations in the MYO7A gene lead to the shaker-1 phenotype in mice that partially replicate the symptoms of Usher syndrome [34]. Developmental and electrophysiological studies have shown that the shaker-1 mice have minor [36] anomalies, such as the predominance of outer hair cells with only two rows of stereocilia instead of the more usual three. These mice showed increased cochlear responses, which is abnormal and may interfere with the cochlear function [36]. In the shaker-1 mutant, the sequence of this loop is changed from NRPM to NPPM This is likely to alter the conformation of the Q helix–loop–R helix structure, which is relatively straight in all myosins. To better understand the physiological changes produced by this mutation, we have performed a detailed study of the ATP hydrolysis mechanism to determine which steps are altered by the R502P shaker-1 mutation

Results

Method

Discussion

Experimental procedures