Structural analysis of the CD2 T lymphocyte antigen by site-directed mutagenesis to introduce a disulphide bond into domain 1

Many proteins have been predicted to contain domains with immunoglobulin-Iikefolds and hence to be members of the immunoglobulin superfamily(IgSF). However, several members lack the Cys residues capableof forming the disulphide bond that forms a characteristic bridgebetween the ß sheets in the Ig fold, e.g. domain 1of the lymphocyte antigen CD2. The assignment of ßstrands in CD2 by sequence analysis was tested by attemptingto introduce a disulphide bridge between the ß sheetsby mutating two residues in the relevant positions to Cys. Mutant,soluble forms of CD2 were expressed in Chinese hamster ovarycell lines and amino add sequencing showed that a disulphidebond was formed as predicted, but not in the control where oneCys residue was misplaced by four residues. Evidence that bothmutated molecules folded correctly is given by the indistinguishablebinding of three monoclonal antibodies recognising differentepftopes on CD2. The 3-D structure of rat CD2 domain 1 has beendetermined by NMR spectroscopy and X-ray crystallography, confirmingthe predictions from the sequence. Applications of this methodof insertion of disulphide residues for probing protein structuresare discussed, together with other structures of IgSF domainslacking the typical inter-sheet disulphide bond.