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-Kb-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 N15s, 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 iN15ΔH and N15sΔH, 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 iN15ΔH and N15sΔH. Both N15sΔH-Fab[H57] and iN15ΔH-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. 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-Kb-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 N15s, 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 iN15ΔH and N15sΔH, 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 iN15ΔH and N15sΔH. Both N15sΔH-Fab[H57] and iN15ΔH-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.