Crystal Structure of Drosophila Unc-45, a Putative Myosin Chaperone

Abstract

UNC-45 is a chaperone that may aid in folding myosin's motor domain. Mutations in UNC-45 cause muscle defects and dysfunction, due to the importance of myosin in muscle structure and contraction. UNC-45 is composed of an N-terminal tetra-tricopeptide (TPR) domain, a C-terminal UCS (UNC-45/Cro1/She4 homology) domain, and a central region that links these. Here we present the crystal structure of Drosophila UNC-45 (dUNC-45), which should serve as a basis for attaining a detailed understanding of its mechanism of action. Bacterially expressed recombinant His-tagged dUNC-45 was purified sequentially using immobilized metal affinity chromatography and size exclusion chromatography. The protein eluted as a single peak, indicating a homogeneous population of protein suitable for crystallization. Crystals were prepared by hanging drop vapor diffusion and x-ray diffraction data were collected to a limit of 3.0 A resolution. For phase determination, a seleno-methionine derivative dUNC-45 crystal was prepared for single wavelength anomalous dispersion (SAD) experiments. Synchrotron data were collected at the Berkeley National Laboratory Advanced Light Source. The diffraction data were processed in HKL2000. Selenium positions were determined and refined in Phenix. The resulting structure was refined against native data using Phaser and maximum-likelihood refinement with Refmac5. Model building was performed in COOT. Our current model has R-cryst and R-free values of 0.24 and 0.28 respectively. The TPR domain is not visible in the model, likely due to flexibility of the domain within the crystal. Overall, the UCS domain is composed of α-helices that form armadillo repeats which stack together to form a supercoiled structure. Based on our structure, it appears that the central and UCS domains make up a single structural unit. Currently, we are pursuing structure-based biochemical assays to pinpoint the protein surfaces that are involved in the binding and chaperone activities of dUNC-45.