Crystallization of a Deglycosylated T Cell Receptor (TCR) Complexed with an Anti-TCR Fab Fragment

• Published in: The Journal of biological chemistry Vol. 271; no. 52; pp. 33639 - 33646
• Main Authors: Liu, Ju, Tse, AlbertG.D., Chang, Hsiu-Ching, Liu, Jin-huan, Wang, Jiahuai, Hussey, Rebecca E., Chishti, Yasmin, Rheinhold, Bruce, Spoerl, Rebecca, Nathenson, Stanley G., Sacchettini, James C., Reinherz, Ellis L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 27.12.1996; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Antibodies, Monoclonal; Baculoviridae; Chromatography, Affinity; Chromatography, Gel; Cricetinae; Crystallization; Crystallography, X-Ray; Glycosylation; Hexosaminidases - metabolism; Immunoglobulin Fab Fragments; Isoelectric Focusing; Leucine Zippers; Mass Spectrometry; Protein Conformation; Receptors, Antigen, T-Cell - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.52.33639

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.