The interaction between the approximately 30 kDa N-terminal domain of enzyme I (EIN) and the approximately 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 15N resonances of EIN upon complex formation by recording a series of 1H-15N correlation spectra of uniformly 15N-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 approximately 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., Seok, 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 alpha 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 alpha and alpha/beta domains. Interestingly, both the backbone and side chain resonances (assigned from a long-range 1H-15N 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 alpha 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.

sugar transport ͉ phosphorylation ͉ x-ray crystallography T he phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) (1) catalyzes the synchronized uptake and phosphorylation of a number of carbohydrates in eubacteria (group translocation) (2, 3). With some variations, the PTS comprises three proteins. In the cytoplasm, PEP phosphorylates enzyme I (EI), which then transfers the phosphoryl group to the histidine phosphocarrier protein, HPr. From HPr, the phosphoryl group is transferred to various sugar-specific membrane associated transporters [enzyme II (EII)], each comprising two cytoplasmic domains, EIIA and EIIB, and an integral membrane domain EIIC. Within EII, EIIA accepts the phosphoryl group from HPr and donates it to EIIB, whereupon EIIC mediates sugar translocation. In addition to controlling sugar translocation, the phosphorylation state of PTS proteins is associated with regulation of metabolic pathways and signaling in bacterial cells (4-8).The Ϸ64-kDa EI is a homodimer, which is more tightly associated at the phosphorylated state than the unphosphorylated state (9-14). The phosphorylation by PEP requires Mg 2ϩ and targets the N atom of His-189 (numbering scheme of EI from Escherichia coli) (15). The dimer association rate constant is two to three orders of magnitude slower than typical rates measured for other dimeric proteins, suggesting that oligomerization is accompanied by major conformational rearrangements (13,16,17). The monomer-dimer equilibrium has been studied in vitro by various methods (18-21), and it has been proposed that the transition plays a regulatory role in the PEP:sugar phosphotransferase system. Yet, transient kinetic studies indicated that the EI dimer phosphorylates HPr without dissociating into monomers (17).Proteolytic cleavage of EI produces two domains (22, 23). The EI N-terminal domain (EIN, residues 1-230) contains the residue that transfers the phosphoryl group, 24) and the HPr-binding domain, whereas the EI C-terminal domain (EIC, residues 261-575) binds PEP in the presence of Mg 2ϩ (the PEP-binding domain) (22,25) and mediates dimerization (26,27). Site-directed mutagenesis showed that Cys-502, located on EIC, is essential for phosphorylation of His-189 by PEP (28). The structure of EIN from E. coli has been determined by x-ray crystallography (29) and NMR spectroscopy (30).

The three-dimensional solution structure of the 259-residue 30 kDa N-terminal domain of enzyme I (EIN) of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been determined by multidimensional nuclear magnetic resonance spectroscopy. Enzyme I, which is autophosphorylated by phosphoenolpyruvate, reversibly phosphorylates the phosphocarrier protein HPr, which in turn phosphorylates a group of membrane-associated proteins, known as enzymes II. To facilitate and confirm NH, 15 N, and 13 C assignments, extensive use was made of perdeuterated 15 N-and 15 N/ 13 Clabeled protein to narrow line widths. Ninety-eight percent of the 1 H, 15 N, and 13 C assignments for the backbone and first side chain atoms of protonated EIN were obtained using a combination of double and triple resonance correlation experiments. The structure determination was based on a total of 4251 experimental NMR restraints, and the precision of the coordinates for the final 50 simulated annealing structures is 0.79 ( 0.18 Å for the backbone atoms and 1.06 ( 0.15 Å for all atoms. The structure is ellipsoidal in shape, approximately 78 Å long and 32 Å wide, and comprises two domains: an R/ domain (residues 1-20 and 148-230) consisting of six strands and three helices and an R-domain (residues 33-143) consisting of four helices. The two domains are connected by two linkers (residues 21-32 and 144-147), and in addition, at the C-terminus there is another helix which serves as a linker between the N-and C-terminal domains of intact enzyme I. A comparison with the recently solved X-ray structure of EIN [Liao, D.-I., Silverton, E., Seok, Y.-J., Lee, B. R., Peterkofsky, A., & Davies, D. R. (1996) Structure 4, 861-872] indicates that there are no significant differences between the solution and crystal structures within the errors of the coordinates. The active site His189 is located in a cleft at the junction of the R and R/ domains and has a pK a of ∼6.3. His189 has a trans conformation about 1 , a g + conformation about 2 , and its N 2 atom accepts a hydrogen bond from the hydroxyl proton of Thr168. Since His189 is thought to be phosphorylated at the N 2 position, its side chain conformation would have to change upon phosphorylation.The phosphoenolpyruvate:sugar phosphotransferase system (PTS), 1 which is found throughout the bacterial kingdom, is responsible for the coupled phosphorylation and translocation of numerous sugars across the cytoplasmic membrane [see Postma et al. (1993) and Herzberg and Klevit (1994) for reviews]. The PTS system comprises a cascade of proteins. The first step in the pathway involves the autophosphorylation of enzyme I (EI) by phosphoenolpyruvate (PEP) to generate phosphorylated EI (P-EI) and pyruvate. P-EI is then responsible for the phosphorylation of a small phosphocarrier protein known as HPr, which in turn phosphorylates a number of membrane-associated proteins, collectively known as enzymes II (EII), which effect the sugar-specific/translocation reactions. EI itself is a 64 kDa pr...