Macdonald Colin J, Tozawa Kaeko, Collins Emily S, Penfold Christopher N, James Richard, Kleanthous Colin, Clayden Nigel J, Moore Geoffrey R
School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK.
J Biomol NMR. 2004 Sep;30(1):81-96. doi: 10.1023/B:JNMR.0000042963.71790.19.
The 61 kDa colicin E9 protein toxin enters the cytoplasm of susceptible cells by interacting with outer membrane and periplasmic helper proteins, and kills them by hydrolysing their DNA. The membrane translocation function is located in the N-terminal domain of the colicin, with a key signal sequence being a pentapeptide region that governs the interaction with the helper protein TolB (the TolB box). Previous NMR studies (Collins et al., 2002 J. Mol. Biol. 318, 787-804) have shown that the N-terminal 83 residues of colicin E9, which includes the TolB box, is largely unstructured and highly flexible. In order to further define the properties of this region we have studied a fusion protein containing residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight-residue linking sequence. 53 of the expected 58 backbone NH resonances for the first 61 residues and all of the expected 7 backbone NH resonances of the linking sequence were assigned with 3D (1)H-(13)C-(15)N NMR experiments, and the backbone dynamics of these regions investigated through measurement of (1)H-(15)N relaxation properties. Reduced spectral density mapping, extended Lipari-Szabo modelling, and fitting backbone R(2) relaxation rates to a polymer dynamics model identifies three clusters of interacting residues, each containing a tryptophan. Each of these clusters is perturbed by TolB binding to the intact colicin, showing that the significant region for TolB binding extends beyond the recognized five amino acids of the TolB box and demonstrating that the binding epitope for TolB involves a considerable degree of order within an otherwise disordered and flexible domain. Abbreviations : Im9, the immunity protein for colicin E9; E9 DNase, the endonuclease domain of colicin E9; HSQC, heteronuclear single quantum coherence; ppm, parts per million; DSS, 2,2-(dimethylsilyl)propanesulfonic acid; TSP, sodium 3-trimethylsilypropionate; T(1 - 61)-DNase fusion protein, residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight residue thrombin cleavage sequence.
61 kDa的大肠杆菌素E9蛋白毒素通过与外膜和周质辅助蛋白相互作用进入敏感细胞的细胞质，并通过水解其DNA将细胞杀死。膜转运功能位于大肠杆菌素的N端结构域，一个关键信号序列是控制与辅助蛋白TolB相互作用的五肽区域（TolB框）。先前的核磁共振研究（Collins等人，《分子生物学杂志》，2002年，第318卷，787 - 804页）表明，大肠杆菌素E9的N端83个残基（包括TolB框）在很大程度上是无结构的且高度灵活。为了进一步确定该区域的特性，我们研究了一种融合蛋白，它包含大肠杆菌素E9的1 - 61位残基，通过一个八残基连接序列与E9 DNase的N端相连。通过三维（1）H - （13）C - （15）N核磁共振实验对前61个残基预期的58个主链NH共振中的53个以及连接序列预期的7个主链NH共振全部进行了归属，并通过测量（1）H - （15）N弛豫特性研究了这些区域的主链动力学。通过简化谱密度映射、扩展的Lipari - Szabo模型以及将主链R(2)弛豫速率拟合到聚合物动力学模型，确定了三个相互作用残基簇，每个簇都包含一个色氨酸。TolB与完整的大肠杆菌素结合会干扰这些簇中的每一个，这表明TolB结合的重要区域超出了公认的TolB框的五个氨基酸，并且证明TolB的结合表位在一个原本无序且灵活的结构域内涉及相当程度的有序性。缩写：Im9，大肠杆菌素E9的免疫蛋白；E9 DNase，大肠杆菌素E9的核酸内切酶结构域；HSQC，异核单量子相干；ppm，百万分之一；DSS，2,2 - （二甲基硅基）丙烷磺酸；TSP，3 - 三甲基硅基丙酸钠；T(1 - 61)-DNase融合蛋白，大肠杆菌素E9的1 - 61位残基通过一个八残基凝血酶切割序列与E9 DNase的N端相连。
