Specificity of Interactions between mDia Isoforms and Rho Proteins
Specificity of Interactions between mDia Isoforms and Rho Proteins
复制标题
DOI：
10.1074/jbc.m805634200
复制
发表时间：
2008-12-12
影响因子：
4.8
通讯作者：
Wittinghofer, Alfred
中科院分区：
文献类型：
--
作者：
Lammers, Michael;Meyer, Simon;Wittinghofer, Alfred
Formins是肌动蛋白成核和聚合的关键调节因子。它们含有双胍同源1 （FH1）和2 （FH2）结构域，作为线状肌动蛋白电缆形成的催化机制。形成蛋白的一个亚类构成了与蝶状体相关的形成蛋白，其成员受Rho亚家族的一个小的gtp结合蛋白的结合调节。这些分子开关蛋白与包括gtpase结合域在内的调节N端媒质(N)结合，导致释放自身抑制。在三种mDia亚型中，mDia1仅被Rho （RhoA， -B和-C）激活，而mDia2和-3也被Rac和Cdc42激活。我们对特异性的决定因素知之甚少。在这里，我们报道了RhoA， Rac1和Cdc42与mDia1和mDia1突变体(mDia（N)-Thr-Ser-His (TSH)）的相互作用，根据结构信息，它应该模仿mDia2和-3。通过生化研究和Cdc42中心点Gpp(NH)p和媒质(N)-TSH复合物的结构分析来分析特异性。mDia1（氨基酸164-166）中的三NNN基序，对应于mDia2/3（氨基酸183-185和190-192）中的TSH基序，以及与Rho插入螺旋相互作用的表位是高亲和力结合所必需的。由于Phe-106的存在，mDia1的三重N基序允许与Rho紧密相互作用，而Rac和Cdc42中相应的His-104与mDia1 /3中的TSH基序形成互补界面。我们还发现，F106H和H104F突变极大地改变了媒质相互作用的亲和力和热力学。
Formins are key regulators of actin nucleation and polymerization. They contain formin homology 1 (FH1) and 2 (FH2) domains as the catalytic machinery for the formation of linear actin cables. A subclass of formins constitutes the Diaphanous-related formins, members of which are regulated by the binding of a small GTP-binding protein of the Rho subfamily. Binding of these molecular switch proteins to the regulatory N-terminal mDia(N), including the GTPase-binding domain, leads to the release of auto-inhibition. From the three mDia isoforms, mDia1 is activated only by Rho (RhoA, -B, and -C), in contrast to mDia2 and -3, which is also activated by Rac and Cdc42. Little is known about the determinants of specificity. Here we report on the interactions of RhoA, Rac1, and Cdc42 with mDia1 and an mDia1 mutant (mDia(N)-Thr-Ser-His (TSH)), which based on structural information should mimic mDia2 and -3. Specificity is analyzed by biochemical studies and a structural analysis of a complex between Cdc42 center dot Gpp(NH)p and mDia(N)-TSH. A triple NNN motif in mDia1 (amino acids 164-166), corresponding to the TSH motif in mDia2/3 (amino acids 183-185 and 190-192), and the epitope interacting with the Rho insert helix are essential for high affinity binding. The triple N motif of mDia1 allows tight interaction with Rho because of the presence of Phe-106, whereas the corresponding His-104 in Rac and Cdc42 forms a complementary interface with the TSH motif in mDia2/3. We also show that the F106H and H104F mutations drastically alter the affinities and thermodynamics of mDia interactions.