A T cell receptor Vα domain expressed in bacteria: Does it dimerize in solution?

Abstract

To evaluate the potential for dimerization through a particular T cell receptor (TCR) domain, we have cloned the cDNA encoding a TCR Vα from a hybridoma with specificity for the human immunodeficiency virus (HIV) envelope glycoprotein 120-derived peptide P18-110 (RGPGRAFVTI) bound to the murine major histocompatibility complex (MHC) class I molecule, H-2D(D). This cDNA was then expressed in a bacterial vector, and protein, as inclusion bodies, was solubilized, refolded, and purified to homogeneity. Yield of the refolded material was from 10 to 50 mg per liter of bacterial culture, the protein was soluble at concentrations as high as 25 mg/ml, and it retained a high level of reactivity with an anti-Vα2 monoclonal antibody. This domain was monomeric both by size exclusion gel chromatography and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Circular dichroism spectra indicated that the folded Vα domains had secondary structure similar to that of single immunoglobulin or TCR domains, consisting largely of β sheet. Conditions for crystallization were established, and at least two crystal geometries were observed; hexagonal bipyramids that failed to diffract beyond ~6 A, and orthorhombic crystals that diffracted to 2.5 A. The dimerization of the Vα domain was investigated further by solution nuclear magnetic resonance spectroscopy, which indicated that dimeric and monomeric forms of the protein were about equally populated at the concentration of 1 mM. Thus, models of TCR-mediated T cell activation that invoke TCR dimerization must consider that some Vα domains have little tendency to form homodimers or multimers.