Switched or not?: the structure of unphosphorylated CheY bound to the N terminus of FliM
Switched or not?: the structure of unphosphorylated CheY bound to the N terminus of FliM
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DOI：
10.1128/jb.00637-06
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发表时间：
2006-11-01
影响因子：
3.2
通讯作者：
Dahlquist, Frederick W.
中科院分区：
文献类型：
--
作者：
Dyer, Collin M.;Dahlquist, Frederick W.
大肠杆菌CheY的磷酸化使其对靶标FliM的亲和力增加20倍。BeF 3-CheY（磷酸化CheY（CheY-P）类似物）与其结合的FliM序列之间的相互作用已在分子详细描述。尽管未磷酸化的CheY与FliM复合时所采用的构象尚不清楚，但有证据表明它与CheY-P的构象相似。为了解决这个问题，我们已经解决了未磷酸化的镁（H）结合的CheY与FliM靶区域（FhM的16个N-末端残基[FliM（16）]）对应的合成肽复合时的晶体结构。虽然肽构象和结合位点与BeF 3--CheY-FliM（16）复合物的那些相似，但无活性的CheY构象在未磷酸化的Mg 2 +-CheY-FliM（16）复合物中大部分保留。之前在生物化学实验中观察到的靶结合位点和磷酸化位点之间的通信是通过保守的侧链相互作用的网络实现的，该网络部分模拟在BeF 3-激活的CheY中观察到的那些。这种结构清楚地表明了β 4-α 4环在Tyr（87）-Tyr（106）偶联机制中发挥的积极作用，该偶联机制使磷酸化位点和靶结合表面之间能够进行变构通信。此外，该结构提供了反应调节蛋白的中间构象的高分辨率视图，其通常被认为是两种状态。
Phosphorylation of Escherichia coli CheY increases its affinity for its target, FliM, 20-fold. The interaction between BeF3--CheY, a phosphorylated CheY (CheY-P) analog, and the FliM sequence that it binds has been described previously in molecular detail. Although the conformation that unphosphorylated CheY adopts in complex with FliM was unknown, some evidence suggested that it is similar to that of CheY-P. To resolve the issue, we have solved the crystallographic structure of unphosphorylated, magnesium (H)-bound CheY in complex with a synthetic peptide corresponding to the target region of FliM (the 16 N-terminal residues of FhM [FliM(16)]). While the peptide conformation and binding site are similar to those of the BeF3--CheY-FliM(16) Complex, the inactive CheY conformation is largely retained in the unphosphorylated Mg2+-CheY-FliM(16) complex. Communication between the target binding site and the phosphorylation site, observed previously in biochemical experiments, is enabled by a network of conserved side chain interactions that partially mimic those observed in BeF3--activated CheY. This structure makes clear the active role that the beta 4-alpha 4 loop plays in the Tyr(87)-Tyr(106) coupling mechanism that enables allosteric communication between the phosphorylation site and the target binding surface. Additionally, this structure provides a high-resolution view of an intermediate conformation of a response regulator protein, which had been generally assumed to be two state.