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Abstract
Myosin is a motor protein that is essential for a variety of processes ranging from intracellular transport to muscle contraction. Folding and assembly of myosin relies on a specific chaperone, UNC-45. To address its substrate-targeting mechanism, we reconstitute the interplay between Caenorhabditis elegans UNC-45 and muscle myosin MHC-B in insect cells. In addition to providing a cellular chaperone assay, the established system enabled us to produce large amounts of functional muscle myosin, as evidenced by a biochemical and structural characterization, and to directly monitor substrate binding to UNC-45. Data from in vitro and cellular chaperone assays, together with crystal structures of binding-deficient UNC-45 mutants, highlight the importance of utilizing a flexible myosin-binding domain. This so-called UCS domain can adopt discrete conformations to efficiently bind and fold substrate. Moreover, our data uncover the molecular basis of temperature-sensitive UNC-45 mutations underlying one of the most prominent motility defects in C. elegans.
Highlights
Myosin is a motor protein that is essential for a variety of processes ranging from intracellular transport to muscle contraction
While the NMY-2 motor domain could be expressed in soluble form, even in the absence of any C. elegans helper chaperone (Fig. 1b), the expression of the MHC-B muscle myosin alone did not yield any soluble recombinant protein, a finding which is consistent with previous reports[21,27]
As it is known that the C. elegans chaperones UNC-45, HSP-1 (Hsp70) and DAF-21 (Hsp90) are critical for myosin folding and assembly[14,19,28], we tested whether co-expression of these chaperones improves the production of the MHC-B motor domain in its soluble form
Summary
Myosin is a motor protein that is essential for a variety of processes ranging from intracellular transport to muscle contraction. To address its substrate-targeting mechanism, we reconstitute the interplay between Caenorhabditis elegans UNC-45 and muscle myosin MHC-B in insect cells. Data from in vitro and cellular chaperone assays, together with crystal structures of binding-deficient UNC-45 mutants, highlight the importance of utilizing a flexible myosin-binding domain. This so-called UCS domain can adopt discrete conformations to efficiently bind and fold substrate. The UNC-45 protein was initially identified in C. elegans, where site-specific temperature-sensitive (ts) mutants revealed the importance of the chaperone for myosin function[10–13]. The recombinant myosin was key to address the basic mechanistic properties of the UNC45 chaperone, revealing for example the molecular basis of ts motility defects of mutant worms harboring point-specific UNC45 mutations. Our data show that these ts mutations affect the myosin-binding capability of UNC-45 rather than its protein stability
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