Mutational Analysis of the Structure and Function of the Chaperoning Domain of UNC-45B.
Mutational Analysis of the Structure and Function of the Chaperoning Domain of UNC-45B.
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UNC-45B 陪伴结构域的结构和功能的突变分析。
DOI：
10.1016/j.bpj.2020.07.012
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发表时间：
2020
影响因子：
3.4
通讯作者：
Oberhauser,AndresF
中科院分区：
文献类型：
--
作者：
Gaziova,Ivana;Moncrief,Taylor;Christian,CourtneyJ;Villarreal,Michael;Powell,Simon;Lee,Hubert;Qadota,Hiroshi;White,MarkA;Benian,GuyM;Oberhauser,AndresF
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 eleganssarcomere structure). Removing the putative client-binding loop altered the secondary structure of the UCS domain (by decreasing theα-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 ofC. eleganssarcomeres. 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.
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DOI：
10.1007/978-1-4939-2522-3_3
发表时间：
2015-01-01
期刊：
STRESS RESPONSES: METHODS AND PROTOCOLS
影响因子：
--
作者：
Haslbeck, Martin;Buchner, Johannes
通讯作者：
Buchner, Johannes
影响因子：
6.8
作者：
Hellerschmied D;Clausen T
通讯作者：
Clausen T
影响因子：
5.6
作者：
Guo, Wei;Chen, Daisi;Epstein, Henry F.
通讯作者：
Epstein, Henry F.
影响因子：
4.8
作者：
Petoukhov, MV;Svergun, DI;Vanoni, MA
通讯作者：
Vanoni, MA
影响因子：
6.1
作者：
Petoukhov MV;Franke D;Shkumatov AV;Tria G;Kikhney AG;Gajda M;Gorba C;Mertens HD;Konarev PV;Svergun DI
通讯作者：
Svergun DI