Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization

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.

De novo design of antibody complementarity determining regions binding a FLAG tetra-peptide

“…3D ). This is lower than the previously measured value of 59.8 ± 0.1 °C for the parental αEE Fab 22 , indicating that the stability of EEf15.4 is lower than that of a typical antibody generated by in vivo somatic hypermutation. Regardless, analytical size exclusion chromatography of the purified Fab peak performed after two months of storage at 4 °C showed that the proteins remained predominantly monomeric, indicting favorable storage stability (Figure S6 ).…”

Tailored placement of a turn-forming PA tag into the structured domain of a protein to probe its conformational state

“…Monoclonal antibodies are ideal candidates as crystallization chaperones, but they have to be individually developed and screened for each target, making the strategy very costly and unpredictable. The idea of developing a portable peptide tag and anti-tag 'chaperoning' antibody has been pursued by Lieberman and colleagues (Johnson et al, 2015), but the technology has not been realized so far. As the inserted PA tag is a 'foreign' structure placed on the client protein, NZ-1 might not have a strong stabilizing effect on the conformation, as in the case of the target-specific chaperone antibody.…”

Approaches to Configuration Determinations of Flexible Marine Natural Products: Advances and Prospects

“…Further, crystallization chaperones can also be used to induce the co-crystallization of target molecules. Crystallization chaperones are molecules that bind covalently or non-covalently to the analyte, helping it to assemble into a crystalline lattice that is then generally applied for protein analysis [ 16 , 17 ]. The Richert group discovered that tetraaryladamantanes (TAAs) are easily co-crystallized with small molecules in the form of inclusive complexes [ 18 ].…”