Structural Study Of Cargo Recogination And Activity Regulation Of Class Ⅴ Myosins

As the representative member of unconventional myosins,class Ⅴ myosins are widely expressed in eukaryotes,from Saccharomyces cerevisiae(Myo2 and Myo4)to vertebrates(MyoⅤa、MyoⅤb and MyoⅤc).The main function of class Ⅴ myosins in cells is to transport cellular components to their destinations,including a broad range of organelles(such as vesicles,mitochondria,melanosomes,etc.),m RNA,etc.,which are involved in various cellular processes,such as cell division,cell migration,endocytosis and exocytosis,and neurotransmitter transmission.Class V myosins can adopt an open/extended and closed/folded conformations.With loaded cargos,Class V myosins have full ATPase activity and can walk along F-actin.Without cargos,the C-terminal globular tail domain folds back to the N-terminal motor domain forming the closed and ATPase activity-inhibited state that loses the motility capacity.There are two important scientific questions in the research field of class Ⅴ myosins: 1)how class Ⅴ myosins specifically recognize a wide range of cargoes through its globular tail domain;2)how it switches from an autoinhibited to active conformation after binding to the cargos.I investigated these two questions in this study.In the part 1,we systematically investigated the mechanism of the globular tail domain recognizing cargos using Myo2,a class Ⅴ myosin protein in Saccharomyces cerevisiae.In order to understand how Myo2-GTD recognizing various cargos,we used Colab Fold,a Alpha Fold2-based complex structure prediction tool,to predict the Myo2-GTD and cargo adaptor complex.First,we use the previously-solved crystal structures of Myo2-GTD and cargo adaptor as a benchmarking to establish the structure prediction scheme of Colab Fold and find a criterion for judging the reliability of the prediction results.Base on this,we successfully predicted the structures of Myo2-GTD in complex with three cargo adapter proteins: Vac17,Kar9,Pea2,which were verified by biochemical experiments.By systematically comparing the interaction details of the complexe structures formed by Myo2 and six cargo adaptors,we summarized the binding modes of three conserved sites on Myo2-GTD,revealing the basis of how Myo2 can recognize and bind multiple cargos specifically.Furthermore,we showed that the Alpha Fold2-based structure prediction can be used to study protein-protein interactions conveniently and efficiently.In the part 2,in order to reveal the mechanisms of the autoinhibition and activation mechanism of class Ⅴ myosins,we used murine MyoⅤa for structural studies.We successfully expressed and purified the complex of the full-length MyoⅤa and calmodulin(CaM)using the insect cell-baculovirus expression system.After screening buffer and cross-linking conditions,we obtained the MyoⅤa-CaM complex with an autoinhibition conformation and determined a 4.78 angstrom cryo-EM structure in the closed state.The Myo Va dimer adopts a triangular-shaped structure with multiple inter-and intra-molecular interactions in establishing the closed state with cargo binding and ATPase activity inhibited.These interactions are formed between the head motor domain and the GTD,the CaM and the coiled-coil,the coiled-coil and the GTD,and the two GTDs.By measuring ATPase activity,we revealed a unique asymmetric autoinhibition mechanism,in which the cargo-binding sites in the two protomers of the Myo Va dimer are differently protected.Thus,our study indicates that the specific and efficient activation of Myo Va requires coincident binding of multiple cargos or cargo adaptors.Together,we obtained the complex structures of Myo2-GTD and cargo adaptors using Colab Fold,the Myo Va full length and calmodulin complex at near-atomic resolution using single-particle cryo-electron microscopy(Cryo-EM),demonstrated the cargo recognition and binding mechanism of class Ⅴ myosins,and revealed the activity regulation mechanism of class Ⅴ myosins mediated by cargo binding.