Strucutre of the UNC-45 Chaperone and its Interaction with Skeletal Muscle Myosin

描述（由申请人提供）：我们建议研究肌球蛋白附属的“UCS结构域”蛋白的结构和作用机制。分子伴侣通过帮助蛋白质折叠和抑制蛋白质变性和聚集在肌肉发育和功能中起关键作用。我们已经证明，β-45是骨骼肌肌球蛋白积累和肌原纤维组装的关键，它与肌球蛋白相互作用，以保护它免受热诱导的聚集。然而，α-45的结构，其与肌球蛋白的相互作用模式及其在骨骼肌疾病中的作用在很大程度上尚未探索。我们的目标是确定果蝇α-45的分子结构，并研究其与肌球蛋白的物理相互作用。我们将测试以下假设：1）ATP结合，增强了β-45分子伴侣的功能，导致蛋白质的结构元件发生构象变化; 2）β-45的UCS结构域与肌球蛋白相互作用。为此，我们将通过结晶、x射线衍射和计算分析，以原子级分辨率研究apo和核苷酸结合态的nitro-45。这将是UCS结构域蛋白的第一个高分辨率结构，UCS结构域蛋白是一类通过哺乳动物在真菌中发现的肌球蛋白相关蛋白。我们还将通过负染色、电子显微镜和单粒子图像分析对与肌球蛋白S-1复合的α-45进行成像，然后将晶体结构对接到类平均投影中。这项研究计划将利用我们在收缩蛋白分析方面的专业知识和高素质合作者的能力。我们的研究将阐明α-45的结构，提供其与ATP相互作用的理解，并定义ATP诱导的构象变化。此外，我们的努力将产生洞察到的肌球蛋白底物，这是至关重要的肌原纤维组装和抗应激的物理相互作用的α-45。公共卫生相关性。β-45分子伴侣对于肌球蛋白（肌肉的分子马达）的功能完整性至关重要。我们将研究β-45分子伴侣的结构及其与肌球蛋白靶点的相互作用。我们的努力将阐明α-45的作用机制，并深入了解它如何在正常肌肉中发挥作用，以及了解它在肌肉应激过程中的作用。

DESCRIPTION (provided by applicant): We propose to study the structure and mechanism of action of the UNC-45 molecular chaperone, a myosin- affiliated "UCS domain" protein. Molecular chaperones play key roles in muscle development and function by aiding protein folding and inhibiting protein denaturation and aggregation. We have demonstrated that UNC-45 is critical for skeletal muscle myosin accumulation and myofibril assembly and that it interacts with myosin to protect it from heat-induced aggregation. However, the structure of UNC-45, its mode of interaction with myosin and its roles in skeletal muscle disease are largely unexplored. Our goal is to define the molecular structure of Drosophila UNC-45 and to study its physical interaction with myosin. We will test the hypotheses that 1) ATP binding, which enhances UNC-45 chaperone function, causes structural elements of the protein to undergo conformational change and 2) the UCS domain of UNC-45 interacts with myosin. To this end, we will examine UNC-45 in the apo- and nucleotide-bound states at atomic-level resolution by crystallization, x-ray diffraction and computational analysis. This will be the first high-resolution structure of a UCS domain protein, a class of myosin-associated proteins found in fungi through mammals. We will also image UNC-45 complexed with myosin S-1 by negative staining, electron microscopy and single particle image analysis followed by docking the crystal structures into class averaged projections. This research program will take advantage of our expertise in contractile protein analysis and the capabilities of highly qualified collaborators. Our studies will elucidate the structure of UNC-45, provide an understanding of its interaction with ATP and define ATP- induced conformational changes. Further, our efforts will yield insight into UNC-45's physical interaction with the myosin substrate, which is critical to myofibril assembly and resistance to stress. PUBLIC HEALTH RELEVANCE. The UNC-45 chaperone is critical for the functional integrity of myosin, the molecular motor of muscle. We will study the structure of the UNC-45 chaperone and its interaction with its myosin target. Our efforts will elucidate the mechanism of action of UNC-45 and provide insight into how it functions in normal muscle as well as an understanding of its role during muscle stress.