Wittekind M, Rajagopal P, Branchini B R, Reizer J, Saier M H, Klevit R E
Department of Biochemistry, University of Washington, Seattle 98195.
Protein Sci. 1992 Oct;1(10):1363-76. doi: 10.1002/pro.5560011016.
The solution structure of the phosphocarrier protein, HPr, from Bacillus subtilis has been determined by analysis of two-dimensional (2D) NMR spectra acquired for the unphosphorylated form of the protein. Inverse-detected 2D (1H-15N) heteronuclear multiple quantum correlation nuclear Overhauser effect (HMQC NOESY) and homonuclear Hartmann-Hahn (HOHAHA) spectra utilizing 15N assignments (reported here) as well as previously published 1H assignments were used to identify cross-peaks that are not resolved in 2D homonuclear 1H spectra. Distance constraints derived from NOESY cross-peaks, hydrogen-bonding patterns derived from 1H-2H exchange experiments, and dihedral angle constraints derived from analysis of coupling constants were used for structure calculations using the variable target function algorithm, DIANA. The calculated models were refined by dynamical simulated annealing using the program X-PLOR. The resulting family of structures has a mean backbone rmsd of 0.63 A (N, C alpha, C', O atoms), excluding the segments containing residues 45-59 and 84-88. The structure is comprised of a four-stranded antiparallel beta-sheet with two antiparallel alpha-helices on one side of the sheet. The active-site His 15 residue serves as the N-cap of alpha-helix A, with its N delta 1 atom pointed toward the solvent to accept the phosphoryl group during the phosphotransfer reaction with enzyme I. The existence of a hydrogen bond between the side-chain oxygen atom of Tyr 37 and the amide proton of Ala 56 is suggested, which may account for the observed stabilization of the region that includes the beta-turn comprised of residues 37-40. If the beta alpha beta beta alpha beta (alpha) folding topology of HPr is considered with the peptide chain polarity reversed, the protein fold is identical to that described for another group of beta alpha beta beta alpha beta proteins that include acylphosphatase and the RNA-binding domains of the U1 snRNP A and hnRNP C proteins.
通过对枯草芽孢杆菌磷酸载体蛋白HPr的未磷酸化形式所采集的二维（2D）核磁共振光谱进行分析，确定了其溶液结构。利用在此报道的¹⁵N化学位移归属以及先前发表的¹H化学位移归属，通过反向检测二维（¹H-¹⁵N）异核多量子相关核Overhauser效应（HMQC NOESY）和同核Hartmann-Hahn（HOHAHA）光谱，来识别在二维同核¹H光谱中未分辨出的交叉峰。从NOESY交叉峰得出的距离约束、从¹H-²H交换实验得出的氢键模式以及从耦合常数分析得出的二面角约束被用于使用可变目标函数算法DIANA进行结构计算。使用X-PLOR程序通过动态模拟退火对计算得到的模型进行优化。所得的结构家族的平均主链均方根偏差为0.63 Å（N、Cα、C′、O原子），不包括含有残基45 - 59和84 - 88的片段。该结构由一个四链反平行β-折叠片组成，在该片的一侧有两个反平行α-螺旋。活性位点的His 15残基作为α-螺旋A的N端帽，其Nδ¹原子指向溶剂，以便在与酶I的磷酸转移反应过程中接受磷酰基。有人提出Tyr 37的侧链氧原子与Ala 56的酰胺质子之间存在氢键，这可能解释了观察到的包括由残基37 - 40组成的β-转角的区域的稳定性。如果将HPr的β-α-β-β-α-β（α）折叠拓扑结构与肽链极性反转的情况一起考虑，该蛋白质折叠与另一组β-α-β-β-α-β蛋白质（包括酰基磷酸酶以及U1 snRNP A和hnRNP C蛋白质的RNA结合结构域）所描述的折叠相同。
