1P4I

Crystal Structure of scFv against peptide GCN4

Summary for 1P4I

• Entry DOI: 10.2210/pdb1p4i/pdb
• Related: 1P4B
• Descriptor: ANTIBODY VARIABLE LIGHT CHAIN, antibody variable heavy chain (3 entities in total)
• Functional Keywords: peptide binder, scfv, picomolar binder, immune system
• Biological source: Mus musculus (house mouse); More
• Total number of polymer chains: 2
• Total formula weight: 26781.28

Authors

Zahnd, C.,Spinelli, S.,Luginbuhl, B.,Jermutus, L.,Amstutz, P.,Cambillau, C.,Pluckthun, A. (deposition date: 2003-04-23, release date: 2004-05-04, Last modification date: 2024-10-09)

Primary citation

Zahnd, C.,Spinelli, S.,Amstutz, P.,Cambillau, C.,Pluckthun, A.
Directed in Vitro Evolution and Crystallographic Analysis of a Peptide-binding Single Chain Antibody Fragment (scFv) with Low Picomolar Affinity.
J.Biol.Chem., 279:18870-18877, 2004

PubMed Abstract: We generated a single chain Fv fragment of an antibody (scFv) with a binding affinity of about 5 pm to a short peptide by applying rigorous directed evolution. Starting from a high affinity peptide binder, originally obtained by ribosome display from a murine library, we generated libraries of mutants with error-prone PCR and DNA shuffling and applied off-rate selection by using ribosome display. Crystallographic analysis of the scFv in its antigen-bound and free state showed that only few mutations, which do not make direct contact to the antigen, lead to a 500-fold affinity improvement over its potential germ line precursor. These results suggest that the affinity optimization of very high affinity binders is achieved by modulating existing interactions via subtle changes in the framework rather than by introducing new contacts. Off-rate selection in combination with ribosome display can evolve binders to the low picomolar affinity range even for peptide targets.

PubMed: 14754898
DOI: 10.1074/jbc.M309169200

Experimental method

X-RAY DIFFRACTION (2.8 Å)