Titin Domains as Mechanical Reporters of UNC-45 Mediated Myosin Folding

Abstract

Myosins are protein machines in which allosteric cross-talk between ATP-binding and hydrolysis and the movement of actin filaments requires precise changes in the polypeptide fold of the motor domain. UNC-45, a member of the UCS family of proteins, acts as a chaperone for myosin and is essential for proper folding and assembly for myosin into muscle thick filaments in vivo. However the molecular mechanisms by which UNC-45 interacts with myosin to promote proper folding of the myosin head domain are not known. We have devised a novel approach to analyze the mechanical refolding of the myosin motor domain at the single molecule level utilizing AFM techniques. By chemically coupling an I27 titin polyprotein to the motor domain of myosin, we introduced a “molecular reporter” into the motor domain, providing a specific attachment point and a well characterized mechanical fingerprint in the AFM measurements. This approach enabled us to study the folding of the motor domain and directly observe the effect of the chaperone UNC-45. The motor domain misfolded after mechanical unfolding, whereas the myosin rod refolded independently of the motor domain. The misfolded motor domain recruited the otherwise robustly folding I27 modules into a misfolded state. Thus, the I27 domains function as a folding sensor. UNC-45 prevents the detrimental interaction between the motor domain and the I27 modules, indicating that it bound to and stabilized the non-native myosin motor domain. Using truncated versions of UNC-45, we localized this protective effect to the C-terminal UCS domain of UNC-45. Our approach enables the study, for the first time, the chaperone effects of UNC-45 on myosin folding in mechanistic detail.This work was funded by NIH grants R01DK073394 (AFO), R01AR050051 (HFE), the Muscular Dystrophy Association and the Cecil and Ida Green Endowment (HFE).