SOLUTION STRUCTURE OF THE 30 KDA N-TERMINAL DOMAIN OF ENZYME-I OF THEESCHERICHIA-COLI PHOSPHOENOLPYRUVATE-SUGAR PHOSPHOTRANSFERASE SYSTEM BY MULTIDIMENSIONAL NMR

The three-dimensional solution structure of the 259-residue 30 kDa N-t erminal domain of enzyme I (EIN) of the phosphoenolpyruvate:sugar phos photransferase system of Escherichia coli has been determined by multi dimensional nuclear magnetic resonance spectroscopy. Enzyme I, which i s 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 an d confirm NH, N-15, and C-13 assignments, extensive use was made of pe rdeuterated N-15- and N-15/C-13-labeled protein to narrow line widths. Ninety-eight percent of the H-1, N-15, and C-13 assignments for the b ackbone and first side chain. atoms of protonated EIN were obtained us ing a combination of double and triple resonance correlation experimen ts. The structure determination was based on a total of 4251 experimen tal NMR restraints, and the precision of the coordinates for the final 50 simulated annealing structures is 0.79 +/- 0.18 Angstrom for the b ackbone atoms and 1.06 +/- 0.15 Angstrom for all atoms. The structure is ellipsoidal in shape, approximately 78 Angstrom long and 32 Angstro m wide, and comprises two domains: an alpha/beta domain (residues 1-20 and 148-230) consisting of six strands and three helices and an alpha -domain (residues 33-143) consisting of four helices. The two domains are connected by two linkers (residues 21-32 and 144-147), and in addi tion, at the C-terminus there is another helix which serves as a linke r between the N- and C-terminal domains of intact enzyme I. A comparis on with the recently solved X-ray structure of EIN [Liao, D.-I., Silve rton, E., Seek, Y.-J., Lee, B. R., Peterkofsky, A., & Davies, D. R. (1 996) Structure 4, 861-872] indicates that there are no significant dif ferences between the solution and crystal structures within the errors of the coordinates. The active site His189 is located in a cleft at t he junction of the a and alpha/beta domains and has a pK(a) of similar to 6.3. His-189 has a trans conformation about chi(1), a g(+) conform ation about chi(2), and its N epsilon 2 atom accepts a hydrogen bond f rom the hydroxyl proton of Thr168. Since His189 is thought to be phosp horylated at the N epsilon 2 position, its side chain conformation wou ld have to change upon phosphorylation.