Audette G F, Engelmann R, Hengstenberg W, Deutscher J, Hayakawa K, Quail J W, Delbaere L T
Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada.
J Mol Biol. 2000 Nov 3;303(4):545-53. doi: 10.1006/jmbi.2000.4166.
The histidine-containing phosphocarrier protein HPr is a central component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), which transfers metabolic carbohydrates across the cell membrane in many bacterial species. In Gram-positive bacteria, phosphorylation of HPr at conserved serine 46 (P-Ser-HPr) plays several regulatory roles within the cell; the major regulatory effect of P-Ser-HPr is its inability to act as a phosphocarrier substrate in the enzyme I reaction of the PTS. In order to investigate the structural nature of HPr regulation by phosphorylation at Ser46, the structure of the P-Ser-HPr from the Gram- positive bacterium Enterococcus faecalis has been determined. X-ray diffraction analysis of P-Ser-HPr crystals provided 10,043 unique reflections, with a 95.1 % completeness of data to 1.9 A resolution. The structure was solved using molecular replacement, with two P-Ser-HPr molecules present in the asymmetric unit. The final R-value and R(Free) are 0.178 and 0.239, respectively. The overall tertiary structure of P-Ser-HPr is that of other HPr structures. However the active site in both P-Ser-HPr molecules was found to be in the "open" conformation. Ala16 of both molecules were observed to be in a state of torsional strain, similar to that seen in the structure of the native HPr from E. faecalis. Regulatory phosphorylation at Ser46 does not induce large structural changes to the HPr molecule. The B-helix was observed to be slightly lengthened as a result of Ser46 phosphorylation. Also, the water mediated Met51-His15 interaction is maintained, again similar to that of the native E. faecalis HPr. The major structural, and thus regulatory, effect of phosphorylation at Ser46 is disruption of the hydrophobic interactions between EI and HPr, in particular the electrostatic repulsion between the phosphoryl group on Ser46 and Glu84 of EI and the prevention of a potential interaction of Met48 with a hydrophobic pocket of EI.
含组氨酸的磷酸载体蛋白HPr是磷酸烯醇丙酮酸：糖磷酸转移酶系统（PTS）的核心组成部分，该系统可在许多细菌物种中跨细胞膜转运代谢性碳水化合物。在革兰氏阳性菌中，保守丝氨酸46位点（P-Ser-HPr）上的HPr磷酸化在细胞内发挥多种调节作用；P-Ser-HPr的主要调节作用是其无法在PTS的酶I反应中作为磷酸载体底物。为了研究丝氨酸46位点磷酸化对HPr调节的结构本质，已确定了革兰氏阳性菌粪肠球菌中P-Ser-HPr的结构。P-Ser-HPr晶体的X射线衍射分析提供了10,043个独立反射，数据完整性达95.1%，分辨率为1.9 Å。该结构通过分子置换法解析，不对称单元中有两个P-Ser-HPr分子。最终的R值和R(Free)分别为0.178和0.239。P-Ser-HPr的整体三级结构与其他HPr结构相同。然而，发现两个P-Ser-HPr分子中的活性位点均处于“开放”构象。观察到两个分子的丙氨酸16处于扭转应变状态，类似于粪肠球菌天然HPr结构中的情况。丝氨酸46位点的调节性磷酸化不会引起HPr分子的大结构变化。观察到由于丝氨酸46磷酸化，β-螺旋略有延长。此外，水介导的甲硫氨酸51-组氨酸15相互作用得以维持，同样类似于粪肠球菌天然HPr。丝氨酸46位点磷酸化的主要结构效应以及由此产生的调节效应是破坏了EI和HPr之间的疏水相互作用，特别是丝氨酸46上的磷酸基团与EI的谷氨酸84之间的静电排斥，以及阻止甲硫氨酸48与EI的疏水口袋发生潜在相互作用。
