Structure and calcium-mediated regulations of unconventional myosin-1c and -VIIa

Abstract

Myosins are actin based molecular motors that function in cell motility and intracellular transportation. Ca²⁺ regulator calmodulin (CaM) acts as light chains for several unconventional myosins, and Ca²⁺ regulates many unconventional myosin-mediated cellular processes. Understanding how Ca²⁺ plays roles in the interaction between CaM and myosins and how these interactions will affect the structure of myosins will be important to understand how myosins function and how they are regulated. The first part of this thesis focuses on the class I myosins. The class I myosins can sense cellular mechanical forces and function as tension-sensitive anchors or transporters. How mechanical load is transduced from the membrane binding tail to the force generating head in myosin-1 is unknown. We determined the crystal structure of the entire tail of mouse myosin-1c in complex with apo-CaM, showing that myosin-1c adopts a stable monomer conformation suited for force transduction. The lever arm helix and the C-terminal extended PH domain of the motor are coupled by a stable post-IQ domain bound to CaM in a highly unusual mode. Ca²⁺ binding to CaM induces major conformational changes in both IQ-motifs and the post-IQ domain and increases flexibility of the myosin-1c tail. Our study provides a structural blueprint for the neck and tail domains of myosin-1 and expands the target binding modes of the master Ca²⁺ signal regulator CaM. The second half of this thesis focuses on another unconventional myosin, Myosin-VIIa. Myosin-VIIa plays important roles in auditory and visual systems. Mutations in the gene myo7a can cause both syndromic (Usher Syndrome) and non-syndromic (DFNA11 and DFNB2) deafness. The structure and function of its highly charged helical region after the IQ motifs (post-IQ) have not been fully studied. We demonstrated that the Myosin-VIIa post-IQ region forms a stable single α helix (SAH) both in crystal and in solution. This SAH together with its preceding IQ5 constitutes a single rigid continuous helix with apo-CaM binding. We discovered that Myosin-VIIa IQ5 can also interact with Ca²⁺-CaM in a mode that is distinct from the IQ5-apo-CaM interaction. This Ca²⁺ induced conformational change breaks the continuity and rigidity of the IQ5-post-IQ helix, serving as a potential regulation mechanism for the Myosin-VIIa force transduction. Integration of IQ5 and SAH into one continuous and stable α-helix not only extends the lengths of the lever arm of but also build the Ca²⁺ regulatory switch into Myosin-VIIa. We proposed that such continuous helical structure composed of the last IQ motif and the following SAH and the corresponding Ca²⁺-regulated structural changes of the lever arm also apply to other unconventional myosins including Myosin-X and Myosin-VI. Moreover, our study of the IQ5-Ca²⁺-CaM interaction, together with several other IQ motifs-Ca²⁺-CaM interactions extend our understandings of a large repertoire of different IQ motifs, which are widely distributed not only in myosins but also in many non-myosin proteins.

Date of Award	2015
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology