Myosins are actin-based molecular motors with diverse cellular and physiological functions, including mechano-sensing, cargo transport, cell migration and organization of actin-based structures such as stereocilia, filopodia and lamella. Despite the large structural and functional diversities among the 38 human myosins, common domain architectures and regulation mechanisms are shared in different myosin superfamily members.

The first half of this thesis focuses on the Myosin-VIIa (Myo7a) lever arm extension rigidity changes in response to calcium signaling. The architecture of the highly charged post-IQ region of Myo7a has been long debated. Combining biochemical studies and crystal structure, our study demonstrates that the post-IQ region of Myo7a indeed forms a stable single α...[ Read more ]

Myosins are actin-based molecular motors with diverse cellular and physiological functions, including mechano-sensing, cargo transport, cell migration and organization of actin-based structures such as stereocilia, filopodia and lamella. Despite the large structural and functional diversities among the 38 human myosins, common domain architectures and regulation mechanisms are shared in different myosin superfamily members.

The first half of this thesis focuses on the Myosin-VIIa (Myo7a) lever arm extension rigidity changes in response to calcium signaling. The architecture of the highly charged post-IQ region of Myo7a has been long debated. Combining biochemical studies and crystal structure, our study demonstrates that the post-IQ region of Myo7a indeed forms a stable single α-helix (SAH). The last IQ motif of Myo7a, IQ5, when bound by apo-CaM, forms a continuous helix of IQ5-SAH in crystal structure, providing evidence that SAH extend the length of lever arm of Myo7a. Upon calcium binding, Ca²⁺-CaM relocates towards the N-terminal region of IQ5, thereby softening the junctional region connecting IQ5 and SAH and altering the lever arm flexibility of Myo7a. My study also characterized the last IQ motifs of other myosin family members that interact with both apo-CaM and Ca²⁺-CaM. Previously identified DFNA11 missense mutations R853C in Myo7a IQ5 region demonstrated disruption of apo-CaM binding and impairment of the motor’s cellular functions.

The second half of this thesis focuses on Myosin-XVIIIa (Myo18a), which contains unique N-terminal PDZ domain for mediating interactions. To elucidate the structural details and regulatory mechanisms of Myo18a in cell migration, we resolved the X-ray structure of Myo18a PDZ domain at 1.8 Å resolution, demonstrating the conventional folding of PDZ domain and the involvement of a conserved helical region at the C-terminus of PDZ domain in stabilizing the PDZ domain for substrate binding. Phosphorylation of a conserved serine residue adjacent to the signature GLGF motif of PDZ domain binding groove was shown to affect Myo18a PDZ domain interaction with its interacting partners Lurap1 and βPIX. Co-transfection of phosphomimetic mutant Myo18a with Lurap1 results in cell spreading phenotype resembling the previous reports of Myo18a knockdown studies. Our study provides evidence of regulation of Myo18a through phosphorylation of its PDZ domain and suggests possible strategy in treatment of cancers with up-regulated Myo18a.