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

The interaction between the similar to 30 kDa N-terminal domain of enz yme I (EIN) and the similar to 9.5 kDa histidine-containing phosphocar rier protein HPr of the Escherichia coli phosphoenolpyruvate:sugar pho sphotransferase system has been investigated by heteronuclear magnetic resonance spectroscopy. The complex is in fast exchange, permitting u s to follow the chemical shift changes of the backbone NH and N-15 res onances 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 o f increasing amounts of HPr at natural isotopic abundance. The equilib rium 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 dissociatio n rate constant is 1100 s(-1). By mapping the backbone chemical shift perturbations on the three-dimensional solution structure of EIN [Garr ett, D. S., Seek, Y.-J., Liao, D.-I., Peterkofsky, A., Gronenborn, A. M., & Clore, G. M. (1997) Biochemistry 36, 2517-2530], we have identif ied the binding surface of EIN in contact with HPr. This surface is pr imarily 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 al so indicate that the active site His 15 of HPr must approach the activ e site His 189 of EIN along the shallow depression at the interface of the cr and alpha/beta domains. Interestingly, both the backbone and s ide 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 i n 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) valu e 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 HP r [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 (re sidues 49-53) of HPr slip between the two pairs of helices constitutin g 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.