Jones B E, Dossonnet V, Küster E, Hillen W, Deutscher J, Klevit R E
University of Washington, Department of Biochemistry and Biomolecular Structure Center, Seattle, Washington 98195-7742, USA.
J Biol Chem. 1997 Oct 17;272(42):26530-5. doi: 10.1074/jbc.272.42.26530.
Catabolite repression of a number of catabolic operons in bacilli is mediated by the catabolite control protein CcpA, the phosphocarrier protein HPr from the phosphoenolpyruvate-dependent sugar transport system (PTS), and a cis-acting DNA sequence termed the catabolite-responsive element (cre). We present evidence that CcpA interacts with HPr that is phosphorylated at Ser46 (Ser(P) HPr) and that these proteins form a specific ternary complex with cre DNA. Titration experiments following the circular dichroism signal of the cre DNA indicate that this complex consists of two molecules of Ser(P) HPr, a CcpA dimer, and the cre sequence. Limited proteolysis experiments indicate that the domain structure of CcpA is similar to other members of the LacI/GalR family of helix-turn-helix proteins, comprised of a helix-turn-helix DNA domain and a C-terminal effector domain. NMR titration of Ser(P) HPr demonstrates that the isolated C-terminal domain of CcpA forms a specific complex with Ser(P) HPr but not with unphosphorylated HPr. Based upon perturbations to the NMR spectrum, we propose that the binding site of the C-terminal domain of CcpA on Ser(P) HPr forms a contiguous surface that encompasses both Ser(P)46 and His15, the site of phosphorylation by enzyme I of the PTS. This allows CcpA to recognize the phosphorylation state of HPr, effectively linking the process of sugar import via the PTS to catabolite repression in bacilli.
芽孢杆菌中多种分解代谢操纵子的分解代谢物阻遏是由分解代谢物控制蛋白CcpA、磷酸烯醇丙酮酸依赖性糖转运系统（PTS）中的磷酸载体蛋白HPr以及一个称为分解代谢物反应元件（cre）的顺式作用DNA序列介导的。我们提供的证据表明，CcpA与在Ser46位点磷酸化的HPr（Ser(P) HPr）相互作用，并且这些蛋白质与cre DNA形成特定的三元复合物。跟踪cre DNA圆二色性信号的滴定实验表明，该复合物由两个Ser(P) HPr分子、一个CcpA二聚体和cre序列组成。有限蛋白酶解实验表明，CcpA的结构域结构与螺旋-转角-螺旋蛋白的LacI/GalR家族的其他成员相似，由一个螺旋-转角-螺旋DNA结构域和一个C端效应结构域组成。Ser(P) HPr的核磁共振滴定表明，分离出的CcpA C端结构域与Ser(P) HPr形成特定复合物，但与未磷酸化的HPr不形成复合物。基于对核磁共振谱的扰动，我们提出CcpA C端结构域在Ser(P) HPr上的结合位点形成一个连续表面，该表面包含Ser(P)46和His15，His15是PTS中酶I的磷酸化位点。这使得CcpA能够识别HPr的磷酸化状态，有效地将通过PTS的糖导入过程与芽孢杆菌中的分解代谢物阻遏联系起来。
