van Nuland N A, Hangyi I W, van Schaik R C, Berendsen H J, van Gunsteren W F, Scheek R M, Robillard G T
Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
J Mol Biol. 1994 Apr 15;237(5):544-59. doi: 10.1006/jmbi.1994.1254.
The solution structure of the histidine-containing phosphocarrier protein HPr from Escherichia coli has been determined by NMR in combination with distance geometry and restrained molecular dynamics. The structure is based on 1520 experimental restraints identified from both three-dimensional 1H-1H-13C and 1H-1H-15N nuclear Overhauser effect multiple-quantum coherence spectroscopy and two-dimensional 1H-1H nuclear Overhauser effect spectra. Thirty-two four-dimensional coordinate frames were produced by metric matrix distance geometry, subjected to distance bounds driven dynamics, projected into three-dimensional space and again subjected to distance-bounds driven dynamics. These 32 distance geometry structures were refined further by restrained molecular dynamics (40 ps) in the GROMOS in vacuo force field. All 32 structures reached acceptable energy minima while satisfying the imposed restraints. Two of these structures were subjected to a further 200 ps of molecular dynamics simulation in water, using time-dependent distance restraining, followed by a 200 ps free simulation without any distance restraining. The resulting structure is very similar to the X-ray structure of Bacillus subtilis HPr, but differs mainly in the position of the two loops containing the active site histidine residue 15 and residues 53 to 57 relative to the rest of the structure. The unfavorable phi torsion angle that was found for residue 16 in the active center of unphosphorylated Streptococcus faecalis HPr was proposed to play a role in the activity of the protein. Although present at the early stages of the structure calculations, this torsion-angle strain disappeared in the final model obtained from molecular dynamics simulations in water using time-averaged distance restraining and upon releasing the distance restraints. This suggests that the strain may be an artifact of crystallization conditions instead of an essential element in the phosphorylation/dephosphorylation process.
通过核磁共振结合距离几何和受限分子动力学方法，已确定了来自大肠杆菌的含组氨酸磷酸载体蛋白HPr的溶液结构。该结构基于从三维1H-1H-13C和1H-1H-15N核Overhauser效应多量子相干光谱以及二维1H-1H核Overhauser效应光谱中识别出的1520个实验约束条件。通过度量矩阵距离几何方法生成了32个四维坐标框架，进行距离边界驱动动力学，投影到三维空间并再次进行距离边界驱动动力学。这32个距离几何结构在GROMOS真空中力场中通过受限分子动力学（40皮秒）进一步优化。所有32个结构在满足施加的约束条件时均达到了可接受的能量最小值。其中两个结构在水中进行了另外200皮秒的分子动力学模拟，采用时间相关距离约束，随后进行200皮秒无任何距离约束的自由模拟。所得结构与枯草芽孢杆菌HPr的X射线结构非常相似，但主要区别在于包含活性位点组氨酸残基15以及残基53至57的两个环相对于结构其余部分的位置。在未磷酸化的粪肠球菌HPr活性中心中发现的残基16的不利φ扭转角被认为在该蛋白的活性中起作用。尽管在结构计算的早期阶段存在这种扭转角应变，但在使用时间平均距离约束并解除距离约束的水中分子动力学模拟获得的最终模型中，这种应变消失了。这表明该应变可能是结晶条件的假象，而不是磷酸化/去磷酸化过程中的必要元素。
