4X0K

Engineered Fab fragment specific for EYMPME (EE) peptide

Summary for 4X0K

• Entry DOI: 10.2210/pdb4x0k/pdb
• Descriptor: Fab fragment heavy chain, Fab fragment light chain (3 entities in total)
• Functional Keywords: antibody fragment, crystallization chaperone, fab fragment, immune system
• Biological source: Homo sapiens (Human); More
• Total number of polymer chains: 4
• Total formula weight: 102892.23

Authors

Johnson, J.L.,Lieberman, R.L. (deposition date: 2014-11-21, release date: 2015-04-08, Last modification date: 2024-11-06)

Primary citation

Johnson, J.L.,Entzminger, K.C.,Hyun, J.,Kalyoncu, S.,Heaner, D.P.,Morales, I.A.,Sheppard, A.,Gumbart, J.C.,Maynard, J.A.,Lieberman, R.L.
Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.
Acta Crystallogr.,Sect.D, 71:896-906, 2015

PubMed Abstract: Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

PubMed: 25849400
DOI: 10.1107/S1399004715001856

Experimental method

X-RAY DIFFRACTION (2.04 Å)