Antibody Variable Domain Interface and Framework Sequence Requirements for Stability and Function by High-Throughput Experiments

• Published in: Structure (London) Vol. 22; no. 1; pp. 22 - 34
• Main Authors: Hsu, Hung-Ju, Lee, Kuo Hao, Jian, Jhih-Wei, Chang, Hung-Ju, Yu, Chung-Ming, Lee, Yu-Ching, Chen, Ing-Chien, Peng, Hung-Pin, Wu, Chih Yuan, Huang, Yu-Feng, Shao, Chih-Yun, Chiu, Kuo Ping, Yang, An-Suei
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 07.01.2014
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - immunology; High-Throughput Nucleotide Sequencing; High-Throughput Screening Assays; Humans; Hydrogen Bonding; Immunoglobulin Variable Region - chemistry; Immunoglobulin Variable Region - immunology; Models, Molecular; Molecular Sequence Data; Peptide Library; Protein Binding; Protein Folding; Protein Stability; Protein Structure, Secondary; Protein Structure, Tertiary; Single-Chain Antibodies - chemistry; Single-Chain Antibodies - immunology; Staphylococcal Protein A - chemistry; Staphylococcal Protein A - immunology; Structure-Activity Relationship; Thermodynamics; Vascular Endothelial Growth Factor A - chemistry; Vascular Endothelial Growth Factor A - immunology
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2013.10.006

Protein structural stability and biological functionality are dictated by the formation of intradomain cores and interdomain interfaces, but the intricate sequence-structure-function interrelationships in the packing of protein cores and interfaces remain difficult to elucidate due to the intractability of enumerating all packing possibilities and assessing the consequences of all the variations. In this work, groups of β strand residues of model antibody variable domains were randomized with saturated mutagenesis and the functional variants were selected for high-throughput sequencing and high-throughput thermal stability measurements. The results show that the sequence preferences of the intradomain hydrophobic core residues are strikingly flexible among hydrophobic residues, implying that these residues are coupled indirectly with antigen binding through energetic stabilization of the protein structures. By contrast, the interdomain interface residues are directly coupled with antigen binding. The interdomain interface should be treated as an integral part of the antigen-binding site. •A few residues in the variable domains are conserved as in consensus sequence•Intradomain core is flexible with alternative combinations of aliphatic side chains•Interdomain interface is conserved in sequence and coupled with antigen binding•The lower cores can be optimized to enhance the variable domains’ stability Hsu et al. determine sequence requirements for antibody variable domain core packing using high-throughput experiments. The results show that only a few residues are conserved as in consensus sequence and that the intradomain core is flexible while the interdomain interface is conserved.