Impacts of Usher syndrome type IB mutations on human myosin VIIa motor function

Abstract

Usher syndrome (USH) is a human hereditary disorder characterized by profound congenital deafness, retinitis pigmentosa, and vestibular dysfunction. Myosin VIIa has been identified as the responsible gene for USH type 1B, and a number of missense mutations have been identified in the affected families. However, the molecular basis of the dysfunction of USH gene, myosin VIIa, in the affected families is unknown to date. Here we clarified the effects of USH1B mutations on human myosin VIIa motor function for the first time. The missense mutations of USH1B significantly inhibited the actin activation of ATPase activity of myosin VIIa. G25R, R212C, A397D, and E450Q mutations abolished the actin-activated ATPase activity completely. P503L mutation increased the basal ATPase activity for 2-3-fold but reduced the actin-activated ATPase activity to 50% of the wild type. While all of the mutations examined, except for R302H, reduced the affinity for actin and the ATP hydrolysis cycling rate, they did not largely decrease the rate of ADP release from actomyosin, suggesting that the mutations reduce the duty ratio of myosin VIIa. Taken together, the results suggest that the mutations responsible for USH1B cause the complete loss of the actin-activated ATPase activity or the reduction of duty ratio of myosin VIIa.

Figure 1. Human myosin VIIa construct

A, Schematic diagram of the full-length human myosin VIIa (HuM7AFull) and the expressed truncated human myosin VIIa construct (HuM7AIQ2). B. SDS-PAGE of the purified wild-type HuM7AIQ2. The HuM7AIQ2 heavy chain was co-expressed with calmodulin (CaM). Lane M; molecular mass markers. C. The molar ratio of co-purified calmodulin light chain determined by densitometry analysis.

Figure 2. Ca²⁺ATPase and the actin-activated Mg²⁺ATPase activity of USH1B mutants

A. Ca²⁺ATPase activity. Ca²⁺ATPase activity was measured in the buffer containing 600 mM KCl, 30 mM Tris-HCl (pH 8.5), 10 mM MgCl₂, 2 mM phosphoenol pyruvate, 20 unit/ml pyruvate kinase, 0.2 mg/ml calmodulin, 20 nM HuM7AIQ2 and 2 mM ATP at 25 °C. B. Basal Ma²⁺-ATPase activity of USH1B mutants. The ATPase activity was measured in the buffer containing 50 mM KCl, 20 mM MOPS-KOH (pH 7.5), 2 mM MgCl₂, 1 mM EGTA, 2 mM phosphoenol pyruvate, 20 unit/ml pyruvate kinase, 0.2 mg/ml calmodulin, 20 nM HuM7AIQ2 and 2 mM MgATP at 25 °C. C. The actin-activated Mg²⁺ATPase activity in the presence of 30 μM actin. Open bars, in the absence of actin; closed bars, in the presence of 30 μM actin. The error bars indicate S.D. for n=3 from three independent preparations.

Figure 3. The actin-dependence of the actin-activated Mg²⁺-ATPase activity of USH1B mutants

A. The actin-activated Mg²⁺-ATPase activity of USH1B mutants showing the actin dependent activation. B. The actin-activated Mg²⁺-ATPase activity of USH1B mutants showing little actin activation. Assay conditions are described in MATERIALS AND METHODS.

Figure 4. ADP dissociation from acto-myosin VIIa USH1B mutants

A. Time course of light-scattering change after mixing acto-HuM7AIQ2/ADP with Mg²⁺-ATP. Measurements were performed in the buffer containing 50 mM KCl, 20 mM MOPS-KOH (pH 7.5), 2 mM MgCl₂, 1 mM EGTA, 0.2 mg/ml calmodulin at 25 °C. Acto-HuM7AIQ2 (0.5 μM HuM7AIQ2 + 0.5 μM actin) in the presence of 0.1 mM Mg²⁺-ADP was mixed with 2 mM Mg²⁺-ATP. The intensity of light-scattering was monitored at 420 nm. The solid line (b) is the best fit to single exponential kinetics with k_obs of 0.56 s⁻1. The line (a) represents the control in the absence of ATP. B. ADP dissociation rates of USH1B mutants. The ADP dissociation rates were measured as described in A for each USH1B mutant. The error bars represent the S.D. from 3 independent experiments.

Figure 5. Actin cosedimentation assay

Actin cosedimentation assays were performed in the buffer containing 50 mM KCl, 20 mM MOPS-KOH (pH 7.5), 2 mM MgCl₂, 1 mM EGTA, 0.2 mg/ml calmodulin. HuM7AIQ2 (0.5 μM) were mixed with 1 mg/ml F-actin in the presence or absence of ATP regeneration system (2 mM phosphoenol pyruvate, 20 unit/ml pyruvate kinase) and 2 mM MgATP, and incubated for 10 min at room temperature. Then, the reaction mixtures were centrifuged at 300,000 X g for 10 min. The supernatants and dissolved pellets were subjected to the densitometry analysis of SDS-PAGE. S, supernatant; P, pellet.

Figure 6. Actin-translocating velocity of USH1B mutants in in vitro motility assay

The movement of the fluorescent-labeled actin filament was observed in 25 mM KCl, 20 mM MOPS-KOH (pH 7.5), 2 mM MgCl₂, 1 mM EGTA, 1 mM dithiothreitol, 36 μg/ml catalase, 4.5 mg/ml glucose, 216 μg/ml glucose oxidase, 0.2 mg/ml calmodulin, and 2 mM MgATP at 25 °C. The error bars indicate S.D. for n=30-40 from three independent preparations.

Figure 7. 3D model for the disruption of the motor activity of human myosin VIIa by the USH1B mutations

A. Crystal structure of the motor domain and first IQ motif. Since the crystal structure of myosin VIIa is not known, the crystal structure of chicken myosin Va motor domain and first IQ motif (49) (1w7j.pdb) is used as a model. Arg212, Arg302, Ala397, and Glu450 are conserved between chicken myosin Va and human myosin VIIa. Pro503 in myosin VIIa is equivalent to Met503 in myosin Va. The side chains of these residues are shown in red (space-filling model). The strut loop region is shown in light blue. B. 3D model structure around the strut loop region. The effect of A397D and P503L mutations on the molecular level were analyzed with the modeling program Swiss-Model (http://swissmodel.expasy.org/) and Swiss-PDB-viewer software. Note that Asp is substituted for Ala at position 397. The strut loop region is shown in light blue. C. 3D model structure around P503 residue. The loop containing P503 of the wild type and P503L mutant are shown in green and pink, respectively.