He Fahu, Wollscheid Hans-Peter, Nowicka Urszula, Biancospino Matteo, Valentini Eleonora, Ehlinger Aaron, Acconcia Filippo, Magistrati Elisa, Polo Simona, Walters Kylie J
Protein Processing Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA.
IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Via Adamello 16, 20139 Milano, Italy.
Cell Rep. 2016 Mar 22;14(11):2683-94. doi: 10.1016/j.celrep.2016.01.079. Epub 2016 Mar 10.
Myosin VI is critical for cargo trafficking and sorting during early endocytosis and autophagosome maturation, and abnormalities in these processes are linked to cancers, neurodegeneration, deafness, and hypertropic cardiomyopathy. We identify a structured domain in myosin VI, myosin VI ubiquitin-binding domain (MyUb), that binds to ubiquitin chains, especially those linked via K63, K11, and K29. Herein, we solve the solution structure of MyUb and MyUb:K63-linked diubiquitin. MyUb folds as a compact helix-turn-helix-like motif and nestles between the ubiquitins of K63-linked diubiquitin, interacting with distinct surfaces of each. A nine-amino-acid extension at the C-terminal helix (Helix2) of MyUb is required for myosin VI interaction with endocytic and autophagic adaptors. Structure-guided mutations revealed that a functional MyUb is necessary for optineurin interaction. In addition, we found that an isoform-specific helix restricts MyUb binding to ubiquitin chains. This work provides fundamental insights into myosin VI interaction with ubiquitinated cargo and functional adaptors.
肌球蛋白VI在早期内吞作用和自噬体成熟过程中的货物运输和分选方面至关重要，而这些过程中的异常与癌症、神经退行性疾病、耳聋和肥厚性心肌病有关。我们在肌球蛋白VI中鉴定出一个结构化结构域，即肌球蛋白VI泛素结合结构域（MyUb），它能结合泛素链，尤其是那些通过K63、K11和K29连接的泛素链。在此，我们解析了MyUb和MyUb:K63连接的双泛素的溶液结构。MyUb折叠成一个紧凑的螺旋-转角-螺旋样基序，并嵌套在K63连接的双泛素的泛素之间，与每个泛素的不同表面相互作用。MyUb的C末端螺旋（螺旋2）上的一个九氨基酸延伸对于肌球蛋白VI与内吞和自噬衔接蛋白的相互作用是必需的。结构导向的突变表明功能性MyUb对于视黄醛结合蛋白相互作用是必要的。此外，我们发现一种异构体特异性螺旋限制了MyUb与泛素链的结合。这项工作为肌球蛋白VI与泛素化货物和功能性衔接蛋白的相互作用提供了基本见解。
