Dataset Information

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.

SUBMITTER: Bujalowski PJ

PROVIDER: S-EPMC4129474 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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UNC-45B chaperone: the role of its domains in the interaction with the myosin motor domain.

Bujalowski Paul J PJ Nicholls Paul P Oberhauser Andres F AF

Biophysical journal 20140801 3

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 fluor ...[more]

PMID: 25099804

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Project description:UNC-45B is a multidomain molecular chaperone that is essential for the proper folding and assembly of myosin into muscle thick filaments in vivo. It has previously been demonstrated that the UCS domain is responsible for the chaperone-like properties of the UNC-45B. To better understand the chaperoning function of the UCS domain of the UNC-45B chaperone, we engineered mutations designed to 1) disrupt chaperone-client interactions by removing and altering the structure of a putative client-interacting loop and 2) disrupt chaperone-client interactions by changing highly conserved residues in a putative client-binding groove. We tested the effect of these mutations by using a, to our knowledge, novel combination of complementary biophysical assays (circular dichroism, chaperone activity, and small-angle x-ray scattering) and in vivo tools (Caenorhabditis elegans sarcomere structure). Removing the putative client-binding loop altered the secondary structure of the UCS domain (by decreasing the α-helix content), leading to a significant change in its solution conformation and a reduced chaperoning function. Additionally, we found that mutating several conserved residues in the putative client-binding groove did not alter the UCS domain secondary structure or structural stability but reduced its chaperoning activity. In vivo, these groove mutations were found to significantly alter the structure and organization of C. elegans sarcomeres. Furthermore, we tested the effect of R805W, a mutation distant from the putative client-binding region, which in humans, has been known to cause congenital and infantile cataracts. Our in vivo data show that, to our surprise, the R805W mutation appeared to have the most drastic detrimental effect on the structure and organization of the worm sarcomeres, indicating a crucial role of R805 in UCS-client interactions. Hence, our experimental approach combining biophysical and biological tools facilitates the study of myosin-chaperone interactions in mechanistic detail.

Dataset Information

UNC-45B chaperone: the role of its domains in the interaction with the myosin motor domain.

Publications

UNC-45B chaperone: the role of its domains in the interaction with the myosin motor domain.

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