Effects of substitutions in the binding surface of an antibody on antigen affinity
Effects of substitutions in the binding surface of an antibody on antigen affinity
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DOI：
10.1093/protein/11.1.65
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发表时间：
1998-01-01
期刊：
影响因子：
--
通讯作者：
Hudson, PJ
中科院分区：
文献类型：
--
作者：
Dougan, DA;Malby, RL;Hudson, PJ
在Fab-神经氨酸酶和scFv-神经氨酸酶复合物的晶体结构中分析了抗体（NC 10）及其抗原流感病毒神经氨酸酶的Fab和单链Fv（scFv）片段之间的相互作用。为了研究抗体-抗原界面中的空腔、盐键和氢键对结合的贡献，通过定点诱变在scFv片段中的6个接触残基处进行14个单个氨基酸置换。用BLAcoreTM光学生物传感器测定每种突变体scFv抗体对神经氨酸酶的结合亲和力。四个突变导致与神经氨酸酶结合的自由能的大的变化（Δ Δ G > 1 kcal/mol），并且一起可能占结合自由能的70%。因此，这些数据支持这样的理论，即少量残基形成“功能表位”，并且对于NC 10与神经氨酸酶的结合是最重要的。已证明神经氨酸酶的抗体残基（Asp）H56和（Lys）N432之间的盐连接对于亲和力很重要，因为用Asn取代（Asp）H56会导致结合自由能大幅降低（Delta Delta G = +2.8 kcal/mol）。由（Tyr）L32和（Asp）H56提供的氢键对于结合也很重要：（Tyr）L32突变为Phe导致结合亲和力显著降低（MG = +1.7 kcal/mol）。疏水相互作用的破坏（货车德瓦尔斯接触）也导致亲和力的显著降低（（Tyr）H99变为Ala，Δ Δ G = +1.5 kcal/mol;（Leu）L94变为Ala，Δ Δ G > +3.0 kcal/mol）。通过用较大的抗体侧链填充界面中的空腔来增加结合亲和力的尝试是不成功的，因为通过新的抗体-抗原相互作用获得的自由能不能补偿空腔结合的水分子的去除。
The interactions between the Fab and single-chain Fv (scFv) fragments of an antibody (NC10) and its antigen, influenza virus neuraminidase, were analysed in the crystal structures of the Fab-neuraminidase and scFv--neuraminidase complexes. To investigate the contribution to binding made by cavities, salt links and hydrogen bonds in the antibody-antigen interface, 14 single amino acid replacements were made at six contact residues in the scFv fragment by site-directed mutagenesis. The binding affinity of each mutant scFv antibody for neuraminidase was determined with a BLAcore(TM) optical biosensor. Four of the mutations resulted in large changes in the free energy of binding to neuraminidase (Delta Delta G > 1 kcal/mol) and together may account for similar to 70% of the free energy of binding. Hence these data support the theory that a small number of residues form the 'functional epitope' and are most important for binding of NC10 to neuraminidase. The salt link between antibody residue (Asp)H56 and (Lys)N432 from neuraminidase was demonstrated to be important for affinity, since substitution of (Asp)H56 with Asn caused a large reduction in the free energy of binding (Delta Delta G = +2.8 kcal/mol). Hydrogen bonds provided by (Tyr)L32 and (Asp)H56 were also important for binding: mutation of (Tyr)L32 to Phe resulted in a significant reduction in binding affinity (MG = +1.7 kcal/mol). Disruption of hydrophobic interactions (van der Waals contacts) led to significant reductions in affinity also ((Tyr)H99 to Ala, Delta Delta G = +1.5 kcal/mol; (Leu)L94 to Ala, Delta Delta G > +3.0 kcal/mol). An attempt to increase binding affinity by filling a cavity in the interface with a larger antibody side chain was unsuccessful, as the free energy gained by new antibody-antigen interactions did not compensate for the removal of cavity-bound water molecules.