PDBsum entry 2a9m

Immune system

PDB id: 2a9m

Contents

Protein chains

233 a.a.

Waters ×315

PDB id:

2a9m

Name:

Immune system

Title:

Structural analysis of a tight-binding fluorescein-scfv; apo form

Structure:

Fluorescein-scfv. Chain: a, b. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Tetramer (from PQS)

Resolution:

2.10Å R-factor: 0.212 R-free: 0.263

Authors:

C.Cambillau,S.Spinelli,A.Honegger,A.Pluckthun

Key ref:

A.Honegger et al. (2005). A mutation designed to alter crystal packing permits structural analysis of a tight-binding fluorescein-scFv complex. Protein Sci, 14, 2537-2549. PubMed id: 16195545 DOI: 10.1110/ps.051520605

Date:

12-Jul-05 Release date: 25-Oct-05

Protein chains

No UniProt id for this chain

Struc: 233 a.a.

DOI no: 10.1110/ps.051520605

Protein Sci 14:2537-2549 (2005)

PubMed id: 16195545

A mutation designed to alter crystal packing permits structural analysis of a tight-binding fluorescein-scFv complex.

A.Honegger, S.Spinelli, C.Cambillau, A.Plückthun.

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.

Selected figure(s)

Figure 1.
Figure 1. Crystal packing and CDR-H3 conformation in the unliganded FITC-E2 scFv. (A) Crystal contacts determine the compact conformation of the long CDR-H3 of FITC-E2, as dominant crystal contacts organize the scFv in the crystal into tightly packed stacks, which pack against each other through smaller crystal contacts. Packing contacts are visualized by color coding the residue according to the fraction of their solvent accessible surface area buried in crystal contacts. Residues that bury >80% of the solvent accessible surface of the free molecule in crystal contacts are colored red; 60%-80%, orange-red; 40%-60%, orange; 20%-40%, yellow-orange; 1%-20%, yellow. Residues not involved in crystal contacts are colored magenta if located within V[L] and blue if located within V[H]. The interface between mol 1 and mol 2, the two molecules in the asymmetric unit, is very similar to the interface between mol 2 and mol 1', an adjacent symmetry related copy of mol 1. (B) The aromatic side chains of Tyr H128 (H100b), Trp H129 (H100c), His H131 (H100e), Phe H132 (H100f), and Tyr H133 (H100g) are located at the core of the dominant crystal interface. Packing of these residues against the bottom of a neighboring molecule determines the conformation of CDR-H3, which alters the shape of the hapten binding pocket. Hapten soaked into the crystals of the unliganded antibody did not localize to the binding pocket, and the wt FITC-E2 failed to crystallize in the presence of the hapten. A mutant scFv in which this dominant crystal contact had been disrupted by the replacement of Trp H129 (H100c) by Ala, a replacement shown not to affect antigen affinity, could be crystallized in the presence of antigen and yielded the structure of the complex.

Figure 4.
Figure 4. Alignment of free and liganded structure and detailed comparison of CDR-H3 conformation. (A) Least squares superposition of V[L] in the unliganded FITC-E2 scFv (blue) and the FITC-E2-hapten complex (red), indicating the difference in the relative orientation of V[L] and V[H] in the two structures. The axis of a 3° rotation relating the V[H] orientations in the freeand the liganded scFv, located in the plain of thefigure, is indicated bya small arrow. (B) CDR-H3 conformation in the unliganded scFv (blue) and the liganded scFv (red). Equivalent atoms are related by green arrows.

The above figures are reprinted by permission from the Protein Society: Protein Sci (2005, 14, 2537-2549) copyright 2005.

Figures were selected by the author.