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

¹ Biochemisches Institut der Universität Zürich, CH-8057 Zürich, Switzerland
² Architecture et Fonction des Macromolécules Biologiques, UPR 9039-CNRS, F-13402 Marseille Cedex 20, France

(RECEIVED April 17, 2005; FINAL REVISION July 15, 2005; ACCEPTED July 20, 2005)

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 Å resolution in the free form and at 3.0 Å 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.

Keywords: structure/function studies; proteins of the immune system; active site; binding site; epitope mapping; crystallography; protein crystallization; forces and stability; mutagenesis (site-directed and general); atomic force microscopy

Abbreviations: V_H, variable domain of antibody heavy chain • V_L, variable domain of antibody light chain • scFv, single-chain variable fragment of an antibody • FITC, fluorescein isothiocyanate • BSA, bovine serum albumin • wt, wild-type • AFM, atomic force microscopy • CDR, complementarity determining region of an antibody • ORE, Oregon Green 488 • PDB, Protein Data Bank

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051520605.

Reprint requests to: Andreas Plückthun, Biochemisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland; e-mail: plueckthun{at}bioc.unizh.ch; fax: 41-44-635-5712.

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				R. Curcio, A. Caflisch, and E. Paci Change of the unbinding mechanism upon a mutation: A molecular dynamics study of an antibody-hapten complex Protein Sci., October 1, 2005; 14(10): 2499 - 2514. [Abstract] [Full Text] [PDF]