Molecular determinants for the recruitment of the ubiquitin-ligase MuRF-1 onto M-line titin
Molecular determinants for the recruitment of the ubiquitin-ligase MuRF-1 onto M-line titin
复制标题
DOI：
10.1096/fj.06-7644com
复制
发表时间：
2007-05-01
期刊：
影响因子：
4.8
通讯作者：
Mayans, Olga
中科院分区：
文献类型：
--
作者：
Mrosek, Michael;Labeit, Dietmar;Mayans, Olga
肌联蛋白在脊椎动物横纹肌中形成肌节内细丝系统，其具有弹性和信号传导特性，因此是机械传导的中心。肌联蛋白在其C-末端附近并直接在激酶结构域之前，含有IG和FnIII模块的保守模式（IG（A168）- IG（A169）-FnIII（A170），由此为A168-A170），其将E3泛素连接酶MuRF-1募集到细丝。这种相互作用被认为调节肌原纤维周转和肌肉的营养状态。我们已经阐明了A168-A170的晶体结构，通过圆二色性（CD）和SEC-MALS表征了MuRF-1变体，并通过等温量热法、SPOTS印迹和下拉测定研究了两种组分的相互作用。这导致了结合的分子决定簇的鉴定。A168-A170示出了延伸的刚性架构，其特征在于跨越其全长的浅表面凹槽和在其中点的明显环突起。在MuRF-1中，C末端螺旋结构域足以以高亲和力结合A168-A170。该螺旋区域可预测地对接到A168-A170的表面凹槽中。此外，下拉分析表明A168-A170中的环突起是MuRF-1识别的关键介导物.我们的研究结果表明，肌联蛋白的这个区域可以作为一个目标，试图治疗抑制MuRF-1介导的肌肉周转，其中小分子结合其独特的结构特征可以阻止MuRF-1的访问。
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 ubiquitinligase 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.