Analysis of binding interactions in an idiotope-antiidiotope protein-protein complex by double mutant cycles
Analysis of binding interactions in an idiotope-antiidiotope protein-protein complex by double mutant cycles
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DOI：
10.1021/bi961769k
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发表时间：
1997-01-07
期刊：
影响因子：
2.9
通讯作者：
Mariuzza, RA
中科院分区：
文献类型：
--
作者：
Goldman, ER;DallAcqua, W;Mariuzza, RA
抗蛋清溶菌酶抗体D1.3和抗D1.3抗体E5.2之间的独特体-抗独特体复合体为研究蛋白-蛋白相互作用提供了一个有用的模型。该复合物的高分辨率晶体结构是可用的[Fields， b.a, Goldbaum, f.a, Ysern， X., Poljak， R. J, Br Mariuzza， R. A. (1995) Nature 374, 739-742]，这两种成分都很容易在大肠杆菌中产生和处理。我们之前分析了D1.3的单个残基对位点定向诱变复合物稳定的相对贡献[Dall'Acqua， W., Goldman， E. R., Eisenstein， E., gr Mariuzza， R. A.（1996）生物化学，35,9667-9676]。在目前的工作中，我们在E5.2与D1.3接触的21个结合位点中的9个位点引入了单丙氨酸取代，发现其中8个位点在配体结合中起重要作用（Delta G（突变型）- Delta G（野生型）> 1.5 kcal/mol）。此外，能量上重要的E5.2和D1.3残基往往在配合物的晶体结构中并置。为了进一步剖析D1.3-E5.2界面中特定相互作用的能量学，我们进行了双突变循环来测量13对氨基酸的偶联，其中9对在晶体结构中直接接触。通过氢键溶剂化作用的侧链的偶能较低（1.3 ~ 1.7 kcal/mol），无论它们是带电-中性对还是中性-中性对，偶能最高（4.3 kcal/mol）。仅形成范德华接触的残基的相互作用能为1.3 ~ 1.6 kcal/mol。晶体结构中不直接接触的远残基之间的循环也显示出显著的耦合（0.5-1.0 kcal/mol）。这些弱的远程相互作用可能是由于溶剂或蛋白质结构的重排或涉及其他残基的二次相互作用。
The idiotope-antiidiotope complex between the anti-hen egg white lysozyme antibody D1.3 and the anti-D1.3 antibody E5.2 provides a useful model for studying protein-protein interactions. A high-resolution crystal structure of the complex is available [Fields, B. A., Goldbaum, F. A., Ysern, X., Poljak, R. J., Br Mariuzza, R. A. (1995) Nature 374, 739-742], and both components are easily produced and manipulated in Escherichia coli. We previously analyzed the relative contributions of individual residues of D1.3 to complex stabilization by site-directed mutagenesis [Dall'Acqua, W., Goldman, E. R., Eisenstein, E., gr Mariuzza, R. A. (1996) Biochemistry, 35, 9667-9676]. In the current work, we introduced single alanine substitutions in 9 out of 21 positions in the combining site of E5.2 involved in contacts with D1.3 and found that 8 of them play a significant role in ligand binding (Delta G(mutant) - Delta G(wild type) > 1.5 kcal/mol). Furthermore, energetically important E5.2 and D1.3 residues tend to be juxtaposed in the crystal structure of the complex. In order to further dissect the energetics of specific interactions in the D1.3-E5.2 interface, double mutant cycles were carried out to measure the coupling of 13 amino acid pairs, 9 of which are in direct contact in the crystal structure. The highest coupling energy (4.3 kcal/mol) was measured for a charged-neutral pair which forms a buried hydrogen bond, while side chains which interact through solvated hydrogen bonds have lower coupling energies (1.3-1.7 kcal/mol), irrespective of whether they involve charged-neutral or neutral-neutral pairs. Interaction energies of similar magnitude (1.3-1.6 kcal/mol) were measured for residues forming only van der Waals contacts. Cycles between distant residues not involved in direct contacts in the crystal structure also showed significant coupling (0.5-1.0 kcal/mol). These weak long-range interactions could be due to rearrangements in solvent or protein structure or to secondary interactions involving other residues.