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...
The alpha/beta subdomain of EIN is topologically similar to the phosphohistidine domain of the enzyme pyruvate phosphate dikinase, which is phosphorylated by PEP on a histidyl residue but does not interact with HPr. It is therefore likely that features of this subdomain are important in the autophosphorylation of enzyme I. The helical subdomain of EIN is not found in pyruvate phosphate dikinase; this subdomain is therefore more likely to be involved in phosphoryl transfer to HPr.
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