Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallizationby Johnson, JL (Johnson, Jennifer L.); Entzminger, KC (Entzminger, Kevin C.); Hyun, J (Hyun, Jeongmin); Kalyoncu, S (Kalyoncu, Sibel); Heaner, DP (Heaner, David P., Jr.); Morales, IA (Morales, Ivan A.); Sheppard, A (Sheppard, Aly); Gumbart, JC (Gumbart, James C.); Maynard, JA (Maynard, Jennifer A.); Lieberman, RL (Lieberman, Raquel L.)
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 angstrom 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 beta-barrel outer membrane protein intimin and beta-helical A2a G protein-coupled receptor (A(2)aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.