Brummell D A, Sharma V P, Anand N N, Bilous D, Dubuc G, Michniewicz J, MacKenzie C R, Sadowska J, Sigurskjold B W, Sinnott B
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Biochemistry. 1993 Feb 2;32(4):1180-7. doi: 10.1021/bi00055a024.
The carbohydrate-binding site in Fab fragments of an antibody specific for Salmonella serogroup B O-polysaccharide has been probed by site-directed mutagenesis using an Escherichia coli expression system. Of the six hypervariable loops, the CDR3 of the heavy chain was selected for exhaustive study because of its significant contribution to binding-site topography. A total of 90 mutants were produced and screened by an affinity electrophoresis/Western blotting method. Those of particular interest were further characterized by enzyme immunoassay, and on this basis seven of the mutant Fabs were selected for thermodynamic characterization by titration microcalorimetry. With regard to residues that hydrogen bond to ligand through backbone interactions, Gly102H could not be substituted, while several side chains could be introduced at Gly100H and Tyr103H with relatively little effect on antigen binding. There was, however, a preference for nonpolar side chains at position 103H. Substitution of His101H with carboxylate and amide side chains gave mutants with binding affinities approaching that of the wild type; complete side-chain removal by mutation to Gly was tolerated with a 10-fold reduction in binding constant. Analysis of binding by titration microcalorimetry revealed some dramatic thermodynamic changes hidden by the similarity of the binding constants. Similar effects were observed with residue changes in an Arg-Asp salt-bridge at the base of the loop. These results indicate that alterations to higher affinity anti-carbohydrate antibodies are characterized by an enthalpy-entropy compensation factor which allows for fundamental changes in the nature of the binding interactions but impedes engineering for increases in affinity.
利用大肠杆菌表达系统，通过定点诱变对沙门氏菌B血清群O-多糖特异性抗体的Fab片段中的碳水化合物结合位点进行了研究。在六个高变环中，由于重链的互补决定区3（CDR3）对结合位点拓扑结构有重要贡献，因此选择对其进行详尽研究。共产生了90个突变体，并通过亲和电泳/蛋白质印迹法进行筛选。通过酶免疫测定对特别感兴趣的突变体进行进一步表征，并在此基础上选择了7个突变Fab片段，通过滴定微量热法进行热力学表征。关于通过主链相互作用与配体形成氢键的残基，Gly102H不能被取代，而在Gly100H和Tyr103H处可以引入几个侧链，对抗原结合的影响相对较小。然而，在103H位置更倾向于非极性侧链。用羧酸盐和酰胺侧链取代His101H得到的突变体，其结合亲和力接近野生型；突变为Gly完全去除侧链时，结合常数降低10倍仍可耐受。通过滴定微量热法分析结合情况发现，结合常数的相似性掩盖了一些显著的热力学变化。在环底部的一个Arg-Asp盐桥中的残基变化也观察到了类似的效应。这些结果表明，高亲和力抗碳水化合物抗体的改变具有焓-熵补偿因子的特征，这允许结合相互作用性质的根本变化，但阻碍了亲和力增加的工程设计。
