While it is known that myosin VIIa is the responsible gene for USH1B, the effect of USH1B mutations on the function of myosin VIIa at the molecular level is unknown. In the present study, we succeeded in isolating human myosin VIIa and clarified, for the first time, how the USH1B mutation hampers the function of myosin VIIa at the molecular level. While the USH1B mutations are distributed in the entire region of the myosin VIIa gene, the majority of the missense mutations are located in the motor domain suggesting that the USH1B phenotype is due to the malfunction of myosin VIIa motor activity. Therefore, we focused our efforts on the missense mutations in the motor domain of myosin VIIa, and examined the effects of these mutations on the motor activity.
One of possible effects of mutation is that the mutation may affect the proper folding and stability of the protein. However, the yields of all USH1B mutant proteins examined in this study were virtually the same as that of the wild-type, suggesting that all mutations examined do not affect the proper folding of myosin VIIa.
In all the functional assays performed in this study, R302H mutation showed little effect on the motor activity of myosin VIIa. It was originally described (43
) that the R302H mutation undergoes difficulty in trying to correlate phenotype/genotype associations because some of the myosin VIIa cDNA clones, isolated from different cDNA libraries, had a histidine at position 302 (26
). Furthermore, Arg302 is not conserved among members of the myosin superfamily, suggesting that Arg302 is not critical for the authentic function of myosin VIIa. Therefore, we concluded that the R302H mutation is not directly responsible for USH1B phenotype.
All other missense mutations tested in this study showed severe dysfunction of the myosin VIIa motor function. The Mg2+-ATPase activities of G25R, R212C, A397D and E450Q mutants were not activated by actin at all, although they had the basal Mg2+-ATPase activity that is similar to the wild-type. These results suggest that these mutations do not hamper the ATP binding and the following ATP hydrolysis, but do impair the transduction pathway between the actin-binding interface and the ATPase active site.
Since these mutants bind to actin in the absence of ATP and dissociate from actin in the presence of ATP, it is anticipated that these mutants can produce the weak actin binding intermediate of myosin VIIa upon ATP binding. We anticipate that the product release of these mutants predominantly takes place without the actin re-binding pathway, thus, no apparent actin activation of the ATPase activity. It is unlikely that these mutants carry the cargo molecules because the majority of the molecules are in the actin-dissociated form in the presence of ATP. On the other hand, P503L mutation showed a two-fold larger basal Mg2+-ATPase activity than wild-type, which was further activated twice by actin. A significant increase in the basal ATPase activity may cause an inefficient energy usage in cells. The ADP release rate of this mutant was higher than the wild-type, suggesting that the duty ratio of this mutant is less than 30%. Therefore, it is less likely that this mutant myosin VIIa can function as a cargo transporter.
Taken together, the present results suggest that USH1B mutations cause either the complete loss of the motor activity of myosin VIIa or the severe reduction of the duty ratio of myosin VIIa. It was shown that Drosophila
myosin VIIa is a high duty ratio motor (29
) and can move procesively when it forms dimer (30
). To date, it is unclear whether human myosin VIIa can form dimer and move processively. On the other hand, it has been suggested that mammalian myosin VIIa serves as a transporter in the pericuticular necklace of the inner ear hair cells (21
) and photoreceptor cells (24
), therefore, it is plausible that human myosin VIIa can move processively, thus functioning as a cargo transporter. This also implies that the processive nature of myosin VIIa is critical for the physiological function of myosin VIIa in the inner ear and retina. The present results are consistent with earlier cell biological findings, and further supports the idea that myosin VIIa functions as a cargo transporter in the cells.
The present results also provide important information for understanding the relationship between the structure and function of myosin VIIa molecule. Of interest is the mutation at Gly25 that is located near the amino terminus of the molecule. Some of the other myosin classes do not contain this region, and the others showed no sequence homology at this region. Nevertheless, G25R mutation completely abolished the actin-translocating activity and the actin activated-ATPase activity. It is plausible that the unique N-terminal domain of myosin VIIa is responsible for the myosin VIIa specific properties of the motor activity, such as a fast actin-attached ATP hydrolysis rate (29
). The importance of the N-terminal region for the myosin motor activity was recently reported for Dictyostelium
myosin II, in which the deletion of the N-terminal region of approximately 80 residues reduced the ATPase and motile activities and the affinities for ADP and actin (13
). Since the primary structure of myosin VIIa at this region is not conserved among myosin family members, it is difficult to predict the structural changes of myosin VIIa at this region by G25R mutation. Further structural studies are required to clarify the mechanism of G25R induced inhibition of myosin VIIa motor activity.
Ala397 is highly conserved among all myosins and lies on the surface of the upper 50 kDa domain in the cleft of the myosin motor domain, which extends from the ATP-binding site to the actin-binding site (). Since structural studies showed that the cleft half closes upon ATP binding, it is thought that the opening and closing of the cleft provide the physical link between the ATP- and actin-binding sites. Consistent with this notion, A397D did not change the basal Ma2+-ATPase activity, but abolished the actin-activated Mg2+-ATPase and motile activities, indicating that Ala397 is essential for the actin dependent process of the ATPase reaction. Based upon the 3D crystal structure of myosin Va, A397 is in close proximity to D570 and L572. The distance between the Oxygen atom of the substituted Asp side chain and the Oxygen atom of the Asp570 side chain is within 3 Å, and it is possible that the substitution of Ala397 to Asp may cause repulsive interaction. The substitution of Ala to Asp may result in steric hindrance with the bulky Leu side chain. Supporting this view, homology modeling suggested that the substitution of Ala397 to Asp pushes the side chain of Asp397 away from Asp570 to increase the distance between the side chain of Asp397 to Asp570 and Leu572 ().
3D model for the disruption of the motor activity of human myosin VIIa by the USH1B mutations
Pro503 is well conserved among the myosin superfamily which lies on the outer surface of the lower domain of myosin (), and this region is thought to be one of the actin-binding sites (14
). Interestingly, the P503L mutation increased the basal Mg2+
-ATPase activity and decreased the actin activation and the actin gliding velocity, suggesting that the P503L mutation hampers the link between the actin binding site and the ATPase active site. Based upon homology modeling, it was predicted that P503L mutation alters the position of the loop A500-I506 (). It is plausible that this conformational change partially mimic the actin bound conformation of myosin.
Arg212 and Glu450 are present in switch I and switch II/relay helix regions, respectively (), which are conserved in all myosins and essential for the myosin motor function. Therefore, it is expected that these mutations would severely impair the motor function. Based upon the structural studies of myosin II, Arg212 is thought to produce a salt bridge with Glu442 in the switch II loop (44
). This salt-bridge is important for the hydrolysis step, because the mutation of Arg212 diminish Pi-burst (45
). The crystal structure of Dictyostelium
myosin II reveals that the replacement of the basic residue at this position prevents the motor domain of myosin to form “closed” conformation that represents a conformational transition of myosin motor domain (48
). The present study of myosin VIIa R212C mutant is consistent with these earlier studies of myosin II, and suggests that the replacement of the basic residue at the position 212 with cysteine is expected to abolish the salt bridge formation, thus hampering the hydrolysis of ATP and destabilizing the formation of “closed” conformation of the motor domain of myosin VIIa.
On the other hand, Glu450 is in close proximity to Lys567, which is in the “strut loop” lying in the position in the cleft connecting the upper and lower 50 kDa domain of the myosin motor domain. Val572 and Asp570 described above are on the same loop (). The distance between the Oxygen atom of the Glu450 and the nitrogen atom of the Lys567 is within 3 Å, which may produce salt bridge. It is plausible that E450Q mutation disrupts the salt-bridge, thus changing the position of the “strut loop” and hampering the ATP hydrolysis cycle of myosin VIIa.
The strut loop is one of the three loops connecting the upper and lower 50K subdomains. The importance of this loop for the myosin motor activity was reported for Dictyostelium
myosin II, in which the insertion or deletion of the residues in this loop abolished strong binding to actin, although the basal ATPase activities were normal (15
). The present results are consistent with the previous results of Dictyostelium
myosin II, and support a notion that A397 or E450 mutation may induce local structural changes at strut loop, thus causing a loss of motor function.
In summary, this is the first report that has clarified the effects of USH1B mutations on the function of the responsible gene product, human myosin VIIa. The USH1B mutations severely hamper the motor function of myosin VIIa. The results indicate that the impairment of the motor function of myosin VIIa is responsible for USH phenotype in humans.