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

Abstract

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...

Tautomeric state and pKa of the phosphorylated active site histidine in the N‐terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate: Sugar phosphotransferase system

“…The pK, of phosphorylated His 189 (-7.3) is one unit higher than that of the unphosphorylated form (-6.3;Garrett et al. 1997a).…”

“…Recently, crystal (Liao et al, 1996) and solution NMR (Garrett et al, 1997a) structures of EIN (1-258 + Arg) have been determined and the interaction surface between EIN and Hpr has been mapped (van Nuland et al, 1995;Garrett et al, 1997b). EIN consists of an (Y domain (residues 33-143) and an alp domain (residues 1-20 and 148-230) connected by linkers (residues 21-32 and 144-147), and a C-terminal helix (residues 233-250) (Fig.…”

“…In contrast, two distinct sets of cross-peak patterns are observed for His 189. One set, corresponding to the unphosphorylated form, displays a pattern of NSl-Hd, Nd-Hd, and N d -H a cross-peaks, with NSl and Ne2 chemical shifts of 191 and 220 ppm, respectively, characteristic of the neutral N61-H tautomeric state (Garrett et al, 1997a). The second set, corresponding to the phosphorylated form, is characterized by a pattern of NS1-Hd, N~2-He1, and NR-HS2 to 241.8 ppm at pH 8.3, close to those expected for the protonated and unprotonated states, respectively, of a nonphosphorylated nitrogen in a phosphoimidazole (Pelton et al, 1993).…”

Active site phosphoryl groups in the biphosphorylated phosphotransferase complex reveal dynamics in a millisecond time scale

“…The imidazole side chain of a histidine adopts either an Ne2-H tautomer or an Nd1-H tautomer in neutral and basic solution, where the Ne2-H tautomer is generally a more stable form [17]. The tautomeric state of the active site His189 of EIN has been well characterized in the unphosphorylated and the phosphorylated states [13,18].…”

Backbone assignment of proteins with known structure using residual dipolar couplings

“…Testing RDC-enhanced assignment was applied to three proteins for which experimental chemical shifts and dipolar couplings have been reported and a highresolution crystal structure is available: ubiquitin (76 aa; PDB codes: 1UBQ [1.8 Å] and 1AAR [2.3 Å]; RDCs: PDB code 1D3ZMR; chemical shifts from TALOS) (Cook et al, 1992;Cornilescu et al, 1998Cornilescu et al, , 1999Vijaykumar et al, 1987), the N-terminal domain of enzyme I of the phosphoenolpyruvate (EIN) (259 aa; PDB code: 1ZYM [2.5 Å]; RDCs: PDB code 3EZAMR; chemical shifts: BMRB code 4106) (Garrett et al, 1997(Garrett et al, , 1999Liao et al, 1996) and two-domain maltose-binding protein (MBP) (370 aa; PDB code: 1DMB [1.8 Å]; RDCs: kindly provided by Lewis Kay; chemical shifts: BMRB code 4354) (Gardner et al, 1998;Mueller et al, 2000;Sharff et al, 1993;Yang et al, 1999). Protons were added to crystal structures using MOLMOL (Koradi et al, 1996).…”