Molecular features of the UNC-45 chaperone critical for binding and folding muscle myosin

Fig. 1. Producing functional myosin in insect cells. a Overview of constructs used for expression studies in insect cells. Western blot analysis of co-expression of NMY-2 (b) or MHC-B (c) with indicated chaperones in Hi5 insect cells. Whole cell lysates (WCL) and soluble fractions (SOL) are shown. 1/3rd of the WCL was applied with respect to the SOL fraction. (SBD–substrate binding domain, NBD–nucleotide binding domain, NTD–N-terminal domain, MD–middle domain, CTD–C-terminal domain). d Pull-down (PD) of Strep-tagged UNC-45 constructs from insect cells. Input samples and elutions were probed with Hsp70, Hsp90, and Strep-tag antibodies. e Upper panel: SEC traces of MHC-B purified from co-expression with the indicated C. elegans chaperones. Lower panel: SDS-PAGE gels showing the elution after NiNTA and indicated SEC fractions

Fig. 2. Effect of UNC-45 mutant proteins on myosin production in insect cells. a UNC-45 mutations that were analyzed in the chaperone assay are shown on the crystal structure of the wild-type protein (PDB code: 4i2z). b Western blot analysis of co-expression of MHC-B with indicated UNC-45 mutant proteins in insect cells. Whole cell lysates (WCL), soluble fractions and elutions after small-scale NiNTA purification are shown for MHC-B. c SEC traces and corresponding SDS-PAGE gels for MHC-B purified from insect cells with and without co-expression of UNC-45. Fractions 1–4 cover the void/aggregate peak, while fractions 5–9 correspond to the elution volume of monomeric functional myosin. d Same analysis as in c for MHC-B co-expressed with UNC-45 UCS domain mutants, TPR domain mutants and ts-mutants

Fig. 3. Characterization of functional MHC-B. a Single ATP turnover experiment monitoring Trp-fluorescence decay (Kobs = 0.073 s−1). b F-actin co-sedimentation assay. In the presence of F-actin, MHC-B is detected in the pellet fraction (P) but is released upon addition of ATP and found in the supernatant (SN). c F-actin-stimulated ATPase activity of MHC-B, yielding Vmax = 0.257 s−1 and KM = 23.1 µM. Error bars represent the s.e.m., of two biological replicates (MHC-B from different purifications), for which three technical replicates have been carried out each. d Stopped-flow analysis measuring dissociation of the actin:MHC-B complex. The inset illustrates example traces of the increase in pyrene fluorescence when 20 µM ATP was mixed with 50 nM pyrene-labelled actin and varying concentrations of MHC-B (5–1000 nM). The increase in fluorescence can be described by an exponential function in which the rate constant is proportional to the ATP concentration and the amplitude of the fluorescence change is proportional to the fractional saturation of actin with MHC-B. The actin affinity derived by this experiment was 10.3 ± 3.7 nM (average of three independent measurements). e Left panel:…

Fig. 4. Interaction of UNC-45 proteins and myosin in vitro. a Analytical SEC and SDS-PAGE analysis of UNC-45 and myosin incubated together at 4 °C or at 27 °C and incubated separately at 27 °C and mixed before the SEC run. Misfolded myosin and bound UNC-45 proteins elute earlier from the SEC column, indicated as high molecular weight fractions (HMW, orange bar). b Controls for interaction studies shown in a. BSA or UNC-45 ΔUCS were incubated with myosin at 27 °C. c Melting temperature as determined by CD spectroscopy for the indicated UNC-45 proteins. d Same analysis as in a for the indicated UNC-45 ts-mutants and myosin, incubated together at 27 °C

Fig. 5. Structural analysis of UNC-45 mutant proteins. a Structural alignment of two different wild-type UNC-45 structures—PDB code: 4i2z, extended UCS domain conformation, PDB code 5mzu, curved UCS conformation. The three helices of each ARM repeat are organized such that helices H3 (colored) form the bottom of the myosin-binding canyon, whereas helices H1 and H2 compose the border of the molecular cleft. Substrates binding to the myosin binding-canyon are illustrated by overlay of ligands co-crystallized with the ARM domain of beta-catenin. b Structural alignment of wild-type UNC-45 (PDB code: 4i2z) and UNC-45 tsG427E—side view (left panel), top view (right panel). The top inset shows the E427 residue overlaid with 2fofc density contoured at 1σ. The bottom inset shows the thermal motion (b) factors displayed on the surface of the tsG427E mutant. N- and C-terminal subdomains of the UCS domain are indicated (green – rigid, magenta – flexible). c Structural alignment of wild-type UNC-45 (PDB code: 4i2z) and tsL822F. Arrows indicate the en bloc movement of the entire UCS domain towards the TPR/central domain. d Structural alignment of wild-type UNC-45 (PDB code: 4i2z) and UNC-45 ΔTPR…

Fig. 6. UNC-45 chaperone function in folding the myosin head domain. Fully functional myosin can be obtained by co-expression with its cognate chaperone UNC-45 in insect cells. UNC-45’s ability to support myosin folding strongly depends on the flexibility of its UCS domain, the functionality of the myosin-binding canyon and its oligomerization properties. UNC-45 ts-mutations limit this conformational flexibility and therefore interfere with myosin binding and chaperone activity