UNC-45B Chaperone: The Role of its Domains in the Interaction with the Myosin Motor Domain

Abstract

The proper folding of many proteins can only be achieved by interaction with molecular chaperones. The molecular chaperone UNC-45B is required for the folding of striated muscle myosin II. However, the precise mechanism by which it contributes to proper folding of the myosin head remains unclear. UNC-45B contains three domains: an N-terminal TPR domain known to bind Hsp90, a Central domain of unknown function, and a C-terminal UCS domain known to interact with the myosin head. Here we used fluorescence titrations methods, dynamic light scattering, and single-molecule atomic force microscopy (AFM) unfolding/refolding techniques to study the interactions of the UCS and Central domains with the myosin motor domain. We found that both the UCS and the Central domains bind to the myosin motor domain. Our data show that the domains bind to distinct subsites on the myosin head, suggesting distinct roles in forming the myosin-UNC-45B complex. To determine the chaperone activity of the UCS and Central domains, we used two different methods: 1), prevention of misfolding using single-molecule AFM, and 2), prevention of aggregation using dynamic light scattering. Using the first method, we found that the UCS domain is sufficient to prevent misfolding of a titin mechanical reporter. Application of the second method showed that the UCS domain but not the Central domain prevents the thermal aggregation of the myosin motor domain. We conclude that while both the UCS and the Central domains bind the myosin head with high affinity, only the UCS domain displays chaperone activity.

Chaperone‐mediated reversible inhibition of the sarcomeric myosin power stroke

“…1c), we estimated the apparent K d of this effect at ∼2.2 μM. This value for the K d is in good agreement with recent fluorescence data from our laboratory [14,25].…”

Interactions between the Dengue Virus Polymerase NS5 and Stem-Loop A

“…Binding curves were fit by using KaleidaGraph software (Synergy Software, PA). In the case of titrations using competition with A(pA) 19 , the relative increase in the fluorescence of the nucleic acid (ΔF) upon binding of DENV NS5 is defined as ΔF obs ϭ (F i Ϫ F 0 )/F 0 , where F i is the fluorescence of the nucleic acid solution at a given titration point, i, and F 0 is the initial fluorescence of the sample (29,(37)(38)(39). In the case of direct titrations of fluorescein-modified SLA, the relative fluorescence change is defined as F i /F 0 .…”

“…The binding constant was determined by using equation 5. In the competition assay, labeled A(pA) 19 (38). Experiments were repeated three times.…”

Thick Filament Protein Network, Functions, and Disease Association

“…While invertebrates express one Unc-45 protein, vertebrates express two, Unc-45a that is present in general cell types and Unc-45b that is specifically expressed in striated muscle cells (453). Extensive biochemical and biophysical work has shown that Unc-45b binds to the myosin head domain via its central and UCS domains (72). Gain and loss of function experiments using in vitro and in vivo models demonstrated that while Unc-45a is critical for cell proliferation and fusion, Unc-45b is essential for myoblast fusion and sarcomeric organization (317, 453, 577, 606).…”