Garrett D S, Seok Y J, Liao D I, Peterkofsky A, Gronenborn A M, Clore G M
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA.
Biochemistry. 1997 Mar 4;36(9):2517-30. 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 A for the backbone atoms and 1.06 +/- 0.15 A for all atoms. The structure is ellipsoidal in shape, approximately 78 A long and 32 A 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 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 alpha and alpha/beta domains and has a pKa of approximately 6.3. His189 has a trans conformation about chi1, a g+ conformation about chi2, and its Nepsilon2 atom accepts a hydrogen bond from the hydroxyl proton of Thr168. Since His189 is thought to be phosphorylated at the N epsilon2 position, its side chain conformation would have to change upon phosphorylation.
糖磷酸转移酶系统中酶I（EIN）259个残基的30 kDa N端结构域的三维溶液结构。酶I被磷酸烯醇丙酮酸自身磷酸化后，可逆地将磷酸载体蛋白HPr磷酸化，而HPr又会将一组与膜相关的蛋白（即酶II）磷酸化。为便于并确认氢、15N和13C的归属，大量使用了全氘代15N和15N/13C标记的蛋白以缩窄谱线宽度。通过双共振和三共振相关实验相结合的方法，获得了质子化EIN主链和首个侧链原子98%的1H、15N和13C归属。结构测定基于总共4251个实验NMR约束条件，最终50个模拟退火结构坐标的精度对于主链原子为0.79±0.18 Å，对于所有原子为1.06±0.15 Å。该结构呈椭圆形，长约78 Å，宽约32 Å，由两个结构域组成：一个α/β结构域（残基1 - 20和148 - 230），由六条链和三个螺旋组成；一个α结构域（残基33 - 143），由四个螺旋组成。这两个结构域通过两个连接子（残基21 - 32和144 - 147）相连，此外，在C端还有另一个螺旋，它作为完整酶I的N端和C端结构域之间的连接子。与最近解析的EIN的X射线结构[廖，D.-I.，西尔弗顿，E.，石，Y.-J.，李，B. R.，彼得科夫斯基，A.，& 戴维斯，D. R.（1996年）《结构》4，861 - 872]进行比较表明，在坐标误差范围内，溶液结构和晶体结构之间没有显著差异。活性位点His189位于α结构域和α/β结构域交界处的一个裂隙中，其pKa约为6.3。His189在χ1处具有反式构象，在χ2处具有g +构象，其Nε2原子接受来自Thr168羟基质子的氢键。由于His189被认为在Nε2位置被磷酸化，其侧链构象在磷酸化时会发生变化。
