Identification by NMR of the binding surface for the histidine-containing phosphocarrier protein HPr on the N-terminal domain of enzyme I of the Escherichia coli phosphotransferase system
Identification by NMR of the binding surface for the histidine-containing phosphocarrier protein HPr on the N-terminal domain of enzyme I of the Escherichia coli phosphotransferase system
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DOI：
10.1021/bi970221q
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发表时间：
1997-04-15
期刊：
影响因子：
2.9
通讯作者：
Gronenborn, AM
中科院分区：
文献类型：
--
作者：
Garrett, DS;Seok, YJ;Gronenborn, AM
本文用异源磁共振波谱法研究了大肠杆菌磷酸烯醇丙酮酸：糖磷酸转移酶系统中类似于30 kDa的酶I N-末端结构域（EIN）和类似于9.5 kDa的含组氨酸的磷酸载体蛋白HPr之间的相互作用。该复合物是在快速交换，使我们能够遵循的骨干NH和N-15共振的EIN后，复杂的化学位移变化;形成通过记录一系列的H-1-N-15相关光谱的均匀N-15标记的EIN在存在下的HPr在天然同位素丰度的增加量。滴定数据分析得出的平衡缔合常数与1.5 x 10（5）M-1相似，解离速率常数的下限为1100 s（-1）。通过将主链化学位移扰动映射到EIN [加勒特，D.美国，寻找，Y。J.，Liao，D.-一、Peterkofsky，A.，Gronenborn，A. M.，& Clore，G. M.（1997）Biochemistry 36，2517-2530]，我们已经鉴定了EIN与HPr接触的结合表面。该表面主要位于ct结构域中，并涉及螺旋H1、H2和H4，以及连接螺旋H2和H2 '的铰链区。这些数据还表明，HPr的活性位点His 15必须沿着Cr和α/β结构域界面处的浅凹陷沿着接近EIN的活性位点His 189。有趣的是，位于α/β结构域中螺旋H6的N-末端的His 189的主链和侧链共振（从长程H-1-N-15相关光谱分配）在复合时仅受到最小的干扰，表明His 189（在不存在磷酸化的情况下）在HPr结合时不经历任何显著的构象变化或pK（α）值变化。根据本研究的结果，以及以前的研究，描绘了相互作用表面的EI HPr [货车Nuland，N。A. J.，博伦斯河舍克河M.，& Robillard，G. T.等人（1995）J. Mol. Biol. 246，180-193]，提出了EIN/HPr复合物的模型，其中HPr的螺旋1（残基16-27）和螺旋环（残基49-53）在构成EIN的ct结构域的两对螺旋之间滑动。此外，我们建议的EIN的螺旋H2和H2'之间的扭结的功能作用，提供了一个灵活的联合这种相互作用发生。
The interaction between the similar to 30 kDa N-terminal domain of enzyme I (EIN) and the similar to 9.5 kDa histidine-containing phosphocarrier protein HPr of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has been investigated by heteronuclear magnetic resonance spectroscopy. The complex is in fast exchange, permitting us to follow the chemical shift changes of the backbone NH and N-15 resonances of EIN upon complex; formation by recording a series of H-1-N-15 correlation spectra of uniformly N-15-labeled EIN in the presence of increasing amounts of HPr at natural isotopic abundance. The equilibrium association constant derived from analysis of the titration data is similar to 1.5 x 10(5) M-1, and the lower limit for the dissociation rate constant is 1100 s(-1). By mapping the backbone chemical shift perturbations on the three-dimensional solution structure of EIN [Garrett, D. S., Seek, Y.-J., Liao, D.-I., Peterkofsky, A., Gronenborn, A. M., & Clore, G. M. (1997) Biochemistry 36, 2517-2530], we have identified the binding surface of EIN in contact with HPr. This surface is primarily located in the ct domain and involves helices H1, H2, and H4, as well as the hinge region connecting helices H2 and H2'. The data also indicate that the active site His 15 of HPr must approach the active site His 189 of EIN along the shallow depression at the interface of the cr and alpha/beta domains. Interestingly, both the backbone and side chain resonances (assigned from a long-range H-1-N-15 correlation spectrum) of His 189, which is located at the N-terminus of helix H6 in he alpha/beta domain, are only minimally perturbed upon complexation, indicating that His 189 (in the absence of phosphorylation) does not undergo any significant conformational change or change in pK(a) value upon HPr binding. On the basis of results of this study, as well as a previous study which delineated the interaction surface for EI on HPr [van Nuland, N. A. J., Boelens, R., Scheek, R. M., & Robillard, G. T. (1995) J. Mol. Biol. 246, 180-193], a model for the EIN/HPr complex is proposed in which helix 1 (residues 16-27) and the helical loop (residues 49-53) of HPr slip between the two pairs of helices constituting the ct domain of EIN. In addition, we suggest a functional role for the kink between helices H2 and H2' of EIN, providing a flexible joint for this interaction to take place.