Solution Structure of the 30 kDa N-Terminal Domain of Enzyme I of the Escherichia coli Phosphoenolpyruvate:Sugar Phosphotransferase System by Multidimensional NMR

• Published in: Biochemistry (Easton) 1997-03, Vol.36 (9), p.2517-2530
• Main Authors: Garrett, Daniel S, Seok, Yeong-Jae, Liao, Der-Ing, Peterkofsky, Alan, Gronenborn, Angela M, Clore, G. Marius
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Crystallography, X-Ray; Escherichia coli; Escherichia coli - enzymology; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Molecular Weight; Phosphoenolpyruvate Sugar Phosphotransferase System - chemistry; Phosphotransferases (Nitrogenous Group Acceptor) - chemistry; Protein Conformation; Solutions
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi962924y

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, 15N, and 13C assignments, extensive use was made of perdeuterated 15N- and 15N/13C-labeled protein to narrow line widths. Ninety-eight percent of the 1H, 15N, and 13C 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 α/β domain (residues 1−20 and 148−230) consisting of six strands and three helices and an α-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 α and α/β domains and has a pK a of ∼6.3. His189 has a trans conformation about χ1, a g+ conformation about χ2, and its Nε2 atom accepts a hydrogen bond from the hydroxyl proton of Thr168. Since His189 is thought to be phosphorylated at the Nε2 position, its side chain conformation would have to change upon phosphorylation.