3ZHD

The crystal structure of single domain antibody 8-4 scaffold.

3ZHD の概要

• エントリーDOI: 10.2210/pdb3zhd/pdb
• 分子名称: MG8-4 SCAFFOLD ANTIBODY, SULFATE ION (3 entities in total)
• 機能のキーワード: immune system, single domain antibody
• 由来する生物種: HOMO SAPIENS (HUMAN)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 27528.25

構造登録者

Song, H.-N.,Woo, E.-J.,Lim, H.-K. (登録日: 2012-12-21, 公開日: 2014-01-08, 最終更新日: 2024-10-23)

主引用文献

Kim, D.,Song, H.,Nam, H.J.,Kim, S.,Park, Y.,Park, J.,Woo, E.,Lim, H.
Directed Evolution of Human Heavy Chain Variable Domain (Vh) Using in Vivo Protein Fitness Filter.
Plos One, 9:98178-, 2014

PubMed Abstract: Human immunoglobulin heavy chain variable domains (VH) are promising scaffolds for antigen binding. However, VH is an unstable and aggregation-prone protein, hindering its use for therapeutic purposes. To evolve the VH domain, we performed in vivo protein solubility selection that linked antibiotic resistance to the protein folding quality control mechanism of the twin-arginine translocation pathway of E. coli. After screening a human germ-line VH library, 95% of the VH proteins obtained were identified as VH3 family members; one VH protein, MG2x1, stood out among separate clones expressing individual VH variants. With further screening of combinatorial framework mutation library of MG2x1, we found a consistent bias toward substitution with tryptophan at the position of 50 and 58 in VH. Comparison of the crystal structures of the VH variants revealed that those substitutions with bulky side chain amino acids filled the cavity in the VH interface between heavy and light chains of the Fab arrangement along with the increased number of hydrogen bonds, decreased solvation energy, and increased negative charge. Accordingly, the engineered VH acquires an increased level of thermodynamic stability, reversible folding, and soluble expression. The library built with the VH variant as a scaffold was qualified as most of VH clones selected randomly were expressed as soluble form in E. coli regardless length of the combinatorial CDR. Furthermore, a non-aggregation feature of the selected VH conferred a free of humoral response in mice, even when administered together with adjuvant. As a result, this selection provides an alternative directed evolution pathway for unstable proteins, which are distinct from conventional methods based on the phage display.

PubMed: 24892548
DOI: 10.1371/JOURNAL.PONE.0098178

実験手法

X-RAY DIFFRACTION (1.962 Å)