Two distinct head-tail interfaces cooperate to suppress activation of vinculin by talin
Two distinct head-tail interfaces cooperate to suppress activation of vinculin by talin
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DOI：
10.1074/jbc.m414704200
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发表时间：
2005-04-29
影响因子：
4.8
通讯作者：
Craig, SW
中科院分区：
文献类型：
--
作者：
Cohen, DM;Chen, H;Craig, SW
纽蛋白被分子内相互作用所抑制，分子内相互作用掩盖了Talin和F-肌动蛋白的结合部位。虽然最近的结构模型仅从纽蛋白尾部(V-t)和残基1-258(D1)之间的相互作用来解释自抑制，但我们发现涉及头部(V-h残基710-836)和V-t的D4结构域的界面是绝对必要的。V-t的电荷-丙氨酸突变揭示了一类位于V-(1-258)结合位点远端的突变体T12和T19，它们在头部结合时的K-d值分别增加了100倍和42倍。接触V-t的D4结构域残基的相互突变产生了一个与T19大小相当的头尾相互作用突变体。这些发现解释了V-t与V-(1-258)结合而不是全长V-h-(1-851)在K-d值上大约120倍的差异。二段自抑制位点对Talin与V-h结合的影响证明了它的重要性。虽然V-t不能与Talin杆状结构域竞争与V-(1-258)结合，但全长V-h很容易发生竞争，这需要D4界面。此外，在完整的纽蛋白中，D4-V-t界面的突变稳定了纽蛋白和Talin杆的结合。在细胞中，这些头尾相互作用的突变体导致局部粘连的肥大和延长。第二个自抑制位点D4-V-t界面的定义支持纽蛋白激活的竞争模型，该模型激活了两个位点上的配体的协同作用。D1-V-t和D4-V-t界面一起提供了在全长纽蛋白中观察到的高亲和力(类似于10(-9))的自抑制。
Vinculin is autoinhibited by an intramolecular interaction that masks binding sites for talin and F-actin. Although a recent structural model explains autoinhibition solely in terms of the interaction between vinculin tail (V-t) and residues 1 - 258 (D1), we find an absolute requirement for an interface involving the D4 domain of head (V-h residues 710 - 836) and V-t. Charge-to-alanine mutations in V-t revealed a class of mutants, T12 and T19, distal to the V-(1-258) binding site, which showed increases in their K-d values for head binding of 100- and 42-fold, respectively. Reciprocal mutation of residues in the D4 domain that contact V-t yielded a head-tail interaction mutant of comparable magnitude to T19. These findings account for the approximately 120-fold difference in K-d values between V-t binding to V-( 1 - 258), as opposed to full-length V-h-( 1 - 851). The significance of a bipartite autoinhibitory site is evidenced by its effects on talin binding to V-h. Whereas V-t fails to compete with the talin rod domain for binding to V-( 1 - 258), competition occurs readily with full-length V-h, and this requires the D4 interface. Moreover in intact vinculin, mutations in the D4-V-t interface stabilize association of vinculin and talin rod. In cells, these head-tail interaction mutants induce hypertrophy and elongation of focal adhesions. Definition of a second autoinhibitory site, the D4-V-t interface, supports the competing model of vinculin activation that invokes cooperative action of ligands at two sites. Together the D1-V-t and D4-V-t interfaces provide the high affinity ( similar to 10(-9)) autoinhibition observed in full-length vinculin.