Structural basis of an engineered dual-specific antibody: conformational diversity leads to a hypervariable loop metal-binding site
Structural basis of an engineered dual-specific antibody: conformational diversity leads to a hypervariable loop metal-binding site
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DOI：
10.1093/protein/gzu033
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发表时间：
2014-10-01
影响因子：
2.4
通讯作者：
Horn, James R.
中科院分区：
文献类型：
--
作者：
Fanning, Sean W.;Walter, Richard;Horn, James R.
为了探索小蛋白界面的双重特异性，我们先前使用组合组氨酸文库方法产生了抗RNaseA VHH抗体的“金属开关”。虽然蛋白质中的大多数金属结合位点位于刚性二级结构中，但含有三个新组氨酸残基的工程VHH抗体(VHHMetal)在柔性高变环中具有金属结合残基。在这里，对VHH金属的自由态和束缚态的晶体结构分析揭示了导致双重功能的结构决定因素。最值得注意的是，当采用金属或RNaseA结合状态时，CDR1以两种不同的构象观察到。此外，诱变研究表明，其中一个工程残基不位于金属结合口袋中，可能是通过影响CDR1的构象而间接促进金属识别的。尽管有这些变化，VHHMetal在与原始抗原RNaseA结合时具有相对较小的能量惩罚(类似于1kcal/mol)，其中工程获得的功能金属结合残基被观察到对RNaseA识别具有有利和不利的贡献。归根结底，独特的金属开关接口体系结构反映了用于生产超高金属的健壮的、基于库的策略。这些结果还表明，即使是小的蛋白质界面，如VHH，也可能在结构和能量上宽容地采用新的功能，同时保持原有的功能。
To explore dual-specificity in a small protein interface, we previously generated a 'metal switch' anti-RNase A VHH antibody using a combinatorial histidine library approach. While most metal-binding sites in proteins are found within rigid secondary structure, the engineered VHH antibody (VHHmetal), which contained three new histidine residues, possessed metal-binding residues within the flexible hypervariable loops. Here, crystal structure analysis of the free and bound states of VHHmetal reveals the structural determinants leading to dual-function. Most notably, CDR1 is observed in two distinct conformations when adopting the metal or RNase A bound states. Furthermore, mutagenesis studies revealed that one of the engineered residues, not located in the metal-binding pocket, contributed indirectly to metal recognition, likely through influencing CDR1 conformation. Despite these changes, VHHmetal possesses a relatively minor energetic penalty toward binding the original antigen, RNase A (similar to 1 kcal/mol), where the engineered gain-of-function metal-binding residues are observed to possess a mix of favorable and unfavorable contributions towards RNase A recognition. Ultimately, the conformationally distinct metal-switch interface architecture reflects the robust, library-based strategy used to produce VHHmetal. These results also suggest that even small protein interfaces, such as VHH, may be structurally and energetically forgiving in adopting novel function, while maintaining original function.