2A9M

Structural Analysis of a Tight-binding Fluorescein-scFv; apo form

2A9M の概要

• エントリーDOI: 10.2210/pdb2a9m/pdb
• 関連するPDBエントリー: 2A9N
• 分子名称: fluorescein-scfv (2 entities in total)
• 機能のキーワード: immunoglobulins, fluorescein, immune system
• 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: 55506.66

構造登録者

Cambillau, C.,Spinelli, S.,Honegger, A.,Pluckthun, A. (登録日: 2005-07-12, 公開日: 2005-10-25, 最終更新日: 2024-11-20)

主引用文献

Honegger, A.,Spinelli, S.,Cambillau, C.,Pluckthun, A.
A mutation designed to alter crystal packing permits structural analysis of a tight-binding fluorescein-scFv complex.
Protein Sci., 14:2537-2549, 2005

PubMed Abstract: The structure of the scFv fragment FITC-E2, obtained from a naive phage antibody scFv library derived from human donors, was determined at 2.1 A resolution in the free form and at 3.0 A in the complexed form. The wild-type (wt) scFv binds fluorescein with a K(D) of 0.75 nM. The free scFv readily crystallizes by compacting its 18 amino acid-long CDR-H3, partially occluding the binding site and further blocking access by binding to the "bottom" of a neighboring scFv molecule with a cluster of exposed aromatic residues within CDR-H3. Only upon mutating one of the residues involved in this dominant crystal contact, an exposed tryptophan in the middle of CDR-H3, crystals of the complex could be obtained. A series of alanine mutants within the putative antigen binding site, covering a range of binding affinities, were used to relate macroscopic thermodynamic and kinetic binding parameters to single-molecule disruption forces measured by AFM. The effects of the mutations on the binding properties, particularly on the fraction of binding-competent molecules within the population, cannot be fully explained by changes in the strength of local interactions. The significant conformational change of CDR-H3 between the free and the liganded form illustrates the plasticity of the binding site. An accompanying study in this issue by Curcio and colleagues presents the molecular dynamics simulation of the forced unbinding experiments and explores possible effects of the mutations on the unbinding pathway of the hapten.

PubMed: 16195545
DOI: 10.1110/ps.051520605

実験手法

X-RAY DIFFRACTION (2.1 Å)