Prasad L, Waygood E B, Lee J S, Delbaere L T
Health Science Building, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada.
J Mol Biol. 1998 Jul 31;280(5):829-45. doi: 10.1006/jmbi.1998.1888.
The tertiary structure of Jel42 Fab fragment complexed with HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli, has been determined at 2.5 A resolution. X-ray diffraction from a larger crystal provided 22,067 unique reflections as compared to 14,763 unique reflections (2.8 A resolution), which were obtained previously from a smaller crystal. The higher resolution allowed for more precise location of amino acid side-chains and for the location of well-ordered water molecules. Five more residues in the Fab fragment are found to be involved in binding HPr and two additional residues are identified as part of the epitope, bringing the totals to 24 and 16, respectively. At least nine water molecules are found at the interface between the two proteins, and these mediate hydrogen bonding interactions between the Fab fragment and HPr. Three additional hydrogen bonds have been identified (bringing the total to ten) and one salt-bridge occurs between LysL50 of the L2 complementarity-determining region (CDR) and GluP66 of HPr. This salt-bridge is the only interaction between HPr and CDRL2; thus all six CDRs are involved in binding. Inspection and empirical energy minimization of mutant HPrs in the complex indicate that, in some cases in the binding interaction, water molecules may compensate for residue alterations. Binding to the mutant SerP64Tyr HPr may require a movement of the HPr main chain. The active centre region of HPr, which is not involved in binding the antibody, and which was not resolved in the 2.8 A resolution structure of the complex, was determined. This active centre determined at pH 5.8, which is completely free of intermolecular contacts due to crystal packing, shows a potential hydrogen bond between the AsnP12 OD1 atom and the HisP15 NE2 atom, and no involvement of the C terminus with HisP15. The HisP15 ND1 atom is the site of phosphorylation in HPr. Although a specific amino acid at residue 12 is not conserved in HPr molecules from all species, a hydrogen bond between the side-chains of residue 12 and HisP15 may be a conserved feature of the active centres.
已在2.5埃分辨率下测定了与HPr复合的Jel42 Fab片段的三级结构。HPr是大肠杆菌磷酸烯醇丙酮酸：糖磷酸转移酶系统的一种磷酸载体蛋白。与之前从小晶体获得的14,763个独立反射（2.8埃分辨率）相比，来自更大晶体的X射线衍射提供了22,067个独立反射。更高的分辨率使得氨基酸侧链的定位更精确，也能确定有序水分子的位置。发现Fab片段中又有5个残基参与结合HPr，另外2个残基被确定为表位的一部分，使得参与结合的残基总数分别达到24个和16个。在两种蛋白质之间的界面处发现至少9个水分子，它们介导了Fab片段与HPr之间的氢键相互作用。又确定了3个额外的氢键（使氢键总数达到10个），并且在L2互补决定区（CDR）的LysL50与HPr的GluP66之间出现了一个盐桥。这个盐桥是HPr与CDRL2之间唯一的相互作用；因此所有6个CDR都参与了结合。对复合物中突变型HPr进行检查和经验性能量最小化表明，在某些结合相互作用的情况下水分子可能补偿残基的改变。与突变型SerP64Tyr HPr的结合可能需要HPr主链的移动。确定了HPr的活性中心区域，该区域不参与与抗体的结合，并且在复合物2.8埃分辨率结构中未解析出来。这个在pH 5.8下确定的活性中心，由于晶体堆积而完全没有分子间接触，显示出AsnP12的OD1原子与HisP15的NE2原子之间存在潜在氢键，并且C末端不与HisP15相关。HisP15的ND1原子是HPr中磷酸化的位点。尽管在所有物种的HPr分子中，第12位残基处的特定氨基酸并不保守，但第12位残基与HisP15侧链之间的氢键可能是活性中心的一个保守特征。
