Studies of the role of phosphorylation and protein binding partners in regulating the function of human class V myosins

Abstract

In this next project we will continue our studies regarding the class V myosins (MyoVs). The combination of structural and functional approaches will be maintained as in the previous project and our goal will be to answer new questions that arose during these two years of work. First, we want to know what the role of phosphorylation in regulating the CBD function of mammalian MyoVs. To this end, we intend to solve the structure of the phospho-mimmetic form of MyoVa-CBD and investigate whether phosphorylation interferes with the auto-inhibition mechanism of this molecular motor and test if it determines the selectivity of partner proteins. Our second question is about the role of alternative splicing in the recognition and selectivity of molecular partners by MyoVs. In this case, we will employ yeast two hybrid assays using as bait constructs containing the MyoVa-CBD fused to different combinations of exons. In parallel, we will perform the same test using as bait constructs of MyoVb and MyoVc similar to those built for MyoVa in order to obtain information regarding redundancy and functional divergence between these three paralogous proteins. To better understand the structural determinants for the recognition of partner proteins by MyoVs, we plan to study complexes between Rab proteins ( Ras -like small GTPases ) 3a, 8a, 10 and 39b and MyoV by X-ray crystallography , SAXS and other techniques that may be necessary. Such interactions have recently been identified in Lindsay et al (Lindsay, et al. 2013). Interestingly, Rab3a and 39b interact with the MyoVa-CBD while Rab8a and Rab10 bind to specific regions of the coiled-coil domain that undergo alternative splicing (Lindsay, et al. 2013). This set of information will have a major contribution to the understanding of structural and functional aspects of the class V myosins. (AU)