Mutational Analysis of the Structure and Function of the Chaperoning Domain of UNC-45B
| Autor: | Guy M. Benian, Courtney J. Christian, Simon Powell, Taylor Moncrief, Hubert Lee, Michael Villarreal, Hiroshi Qadota, Andres F. Oberhauser, Ivana Gaziova, Mark A. White |
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| Rok vydání: | 2020 |
| Předmět: |
Sarcomeres
Circular dichroism Biophysics Myosins Sarcomere 03 medical and health sciences 0302 clinical medicine Myosin Animals Humans Caenorhabditis elegans Caenorhabditis elegans Proteins Protein secondary structure 030304 developmental biology 0303 health sciences biology Chemistry Articles biology.organism_classification Structure and function Cell biology Mutational analysis Chaperone (protein) biology.protein 030217 neurology & neurosurgery Molecular Chaperones |
| Zdroj: | Biophys J |
| ISSN: | 0006-3495 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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