Mrosek Michael, Labeit Dietmar, Witt Stephanie, Heerklotz Heiko, von Castelmur Eleonore, Labeit Siegfried, Mayans Olga
Division of Structural Biology, Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland.
FASEB J. 2007 May;21(7):1383-92. doi: 10.1096/fj.06-7644com. Epub 2007 Jan 10.
Titin forms an intrasarcomeric filament system in vertebrate striated muscle, which has elastic and signaling properties and is thereby central to mechanotransduction. Near its C-terminus and directly preceding a kinase domain, titin contains a conserved pattern of Ig and FnIII modules (Ig(A168)-Ig(A169)-FnIII(A170), hereby A168-A170) that recruits the E3 ubiquitin-ligase MuRF-1 to the filament. This interaction is thought to regulate myofibril turnover and the trophic state of muscle. We have elucidated the crystal structure of A168-A170, characterized MuRF-1 variants by circular dichroism (CD) and SEC-MALS, and studied the interaction of both components by isothermal calorimetry, SPOTS blots, and pull-down assays. This has led to the identification of the molecular determinants of the binding. A168-A170 shows an extended, rigid architecture, which is characterized by a shallow surface groove that spans its full length and a distinct loop protrusion in its middle point. In MuRF-1, a C-terminal helical domain is sufficient to bind A168-A170 with high affinity. This helical region predictably docks into the surface groove of A168-A170. Furthermore, pull-down assays demonstrate that the loop protrusion in A168-A170 is a key mediator of MuRF-1 recognition. Our findings indicate that this region of titin could serve as a target to attempt therapeutic inhibition of MuRF-1-mediated muscle turnover, where binding of small molecules to its distinctive structural features could block MuRF-1 access.
肌联蛋白在脊椎动物横纹肌中形成肌节内细丝系统，该系统具有弹性和信号传导特性，因此是机械转导的核心。在肌联蛋白的C末端附近且紧接激酶结构域之前，它包含Ig和FnIII模块的保守模式（Ig(A168)-Ig(A169)-FnIII(A170)，以下简称A168 - A170），该模式可将E3泛素连接酶MuRF - 1募集到细丝上。这种相互作用被认为可调节肌原纤维更新和肌肉的营养状态。我们解析了A168 - A170的晶体结构，通过圆二色性（CD）和尺寸排阻色谱 - 多角度激光散射（SEC - MALS）对MuRF - 1变体进行了表征，并通过等温滴定量热法、斑点印迹法和下拉实验研究了这两种成分的相互作用。这使得我们能够确定结合的分子决定因素。A168 - A170呈现出一种延伸的、刚性的结构，其特征在于一个贯穿全长的浅表面凹槽以及在中点处明显的环突出。在MuRF - 1中，一个C末端螺旋结构域足以与A168 - A170高亲和力结合。这个螺旋区域可预测地对接至A168 - A170的表面凹槽中。此外，下拉实验表明A168 - A170中的环突出是MuRF - 1识别的关键介导因子。我们的研究结果表明，肌联蛋白的这一区域可作为尝试治疗性抑制MuRF - 1介导的肌肉更新的靶点，小分子与其独特结构特征的结合可能会阻止MuRF - 1的作用。
