Myo1c mutations associated with hearing loss cause defects in the interaction with nucleotide and actin

Abstract

Three heterozygous missense mutations in the motor domain of myosin 1c (Myo1c), which mediates adaptation in the inner ear, are associated with bilateral sensorineural hearing loss in humans. With transient kinetic analyses, steady-state ATPase and motility assays, and homology modeling, we studied the interaction of these mutants with nucleotide and actin using a truncated construct, Myo1c(1IQ-SAH), which includes an artificial lever arm. Results indicate that mutation R156W, near switch 1, affects the nucleotide-binding pocket and the calcium binding by disrupting switch 1 movement. Mutation V252A, in the K helix of the upper 50 kDa domain, showed reduced actin affinity consistent with disruption of communication between the actin- and nucleotide-binding sites. T380M, in a Myo1c-specific insert in the HO linker, displayed aberrant changes in most kinetic parameters and uncoupling of the ATPase from motility. These data allow for an interpretation of how these mutations might affect adaptation.

Scheme 1

M myosin, T ATP, D ADP, Pi inorganic phosphate, asterisks indicate a quench in fluorescence

Scheme 2

A actin, M myosin, A·M actomyosin complexes, T ATP, D ADP, Pi inorganic phosphate, rate constants denoted with ′ were determined in the presence of actin

Fig. 1a–d

Actin-activated Mg²⁺-ATPase activity. The Mg²⁺-ATPase activity of a Myo1c^1IQ-SAH-WT, b Myo1c^1IQ-SAH-R156W, c Myo1c^1IQ-SAH-V252A, and d Myo1c^1IQSAH-T380M was measured at 37°C and actin concentrations of 0–75 μM in the presence (pCa 4.6) and absence (pCa 8.9) of calcium

Fig. 2a–c

Transient kinetic parameters determined by stopped-flow spectroscopy for hearing mutants. Displayed are values for a Myo1c^1IQ-SAH-R156W, b Myo1c^1IQ-SAH-V252A, and c Myo1c^1IQ-SAH-T380M, relative to those obtained for Myo1c^1IQ-SAH-WT. A value of 1.0, indicated by the solid horizontal line, indicates that the two values are equal. The dashed lines represent 30% deviation from 1.0. Left EGTA, right calcium

Fig. 3

Homology model of the crystal structure of the Myo1c motor domain showing the positions of three residues that are associated with hearing disorders when mutated. The regions around each of the three residues are enlarged and rotated for the best view (right). The model was created with Swiss-Model [32] using the solved crystal structure of Dictyotelium MyoE (ADP.vanadate structure) as a template (PDB 1LKX-A). Color-coded structural elements: magenta switch 1 and switch 2, orange P-loop, green SW-2 helix, sky blue β-stand 5, red helix G and loop 1, yellow helix O and HO-linker, dark pink myopathy loop. Bound nucleotide (ADP) and point mutations are shown in space-fill mode