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

TitleStructural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.
Publication TypeJournal Article
Year of Publication2015
AuthorsJohnson, JL, Entzminger, KC, Hyun, J, Kalyoncu, S, Heaner, DP, Morales, IA, Sheppard, A, Gumbart, JC, Maynard, JA, Lieberman, RL
JournalActa Crystallogr D Biol Crystallogr
Volume71
IssuePt 4
Pagination896-906
Date Published2015 Apr
ISSN1399-0047
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.

DOI10.1107/S1399004715001856
Alternate JournalActa Crystallogr. D Biol. Crystallogr.
PubMed ID25849400
PubMed Central IDPMC4388267
Grant ListDK091357 / DK / NIDDK NIH HHS / United States
GM095638 / GM / NIGMS NIH HHS / United States
K22-AI100927 / AI / NIAID NIH HHS / United States
R01 GM095638 / GM / NIGMS NIH HHS / United States