Crystallization of a deglycosylated T cell receptor (TCR) complexed with an anti-TCR Fab fragment

Abstract

A strategy to overexpress T cell receptors (TCRs) in Lec3.2.8.1 cells has been developed using the 'Velcro' leucine zipper sequence to facilitate α-β pairing. Upon secretion in culture media, the VSV-8-specific/H2-K(b)- restricted N15 TCR could be readily immunopurified using the anti-leucine zipper monoclonal antibody 2H11, with a yield of 5-10 mg/liter. Mass spectrometry analysis revealed that all attached glycans were GlcNAc2- Man5. Following Superdex 200 gel filtration to remove aggregates, wild-type N15 or N158, a C183S variant lacking the unpaired cysteine at amino acid residue 183 in the Cβ domain, was thrombin-cleaved and endoglycosidase H- digested, and the two derivatives were termed iN15Δ(H) and N158Δ(H) respectively, and sized by Superdex 75 chromatography to high purity. N- terminal and C-terminal microsequencing analysis showed the expected unique termini of N15 α and β subunits. Nevertheless, neither protein crystallized under a wide range of conditions. Subsequently, we produced a Fab fragment of the murine TCR Cβ-specific hamster monoclonal antibody H57 and complexed the Fab fragment with iN15Δ(H) and N158Δ(H). Both N158Δ(H)-Fab[H57] and iN15Δ(H)-Fab[H57] complexes crystallize, with the former diffracting to 2.8- Å resolution. These findings show that neither intact glycans nor the conserved and partially exposed Cys-183 is required for protein stability. Furthermore, our results suggest that the H57 Fab fragment aids in the crystallization of TCRs by altering their molecular surface and/or stabilizing inherent conformational mobility.