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-like folds and hence to be members of the immunoglobulin superfamily (IgSF). However, several members lack the Cys residues capable of forming the disulphide bond that forms a characteristic bridge between the beta sheets in the Ig fold, e.g. domain 1 of the lymphocyte antigen CD2. The assignment of beta strands in CD2 by sequence analysis was tested by attempting to introduce a disulphide bridge between the beta sheets by mutating two residues in the relevant positions to Cys. Mutant, soluble forms of CD2 were expressed in Chinese hamster ovary cell lines and amino acid sequencing showed that a disulphide bond was formed as predicted, but not in the control where one Cys residue was misplaced by four residues. Evidence that both mutated molecules folded correctly is given by the indistinguishable binding of three monoclonal antibodies recognising different epitopes on CD2. The 3-D structure of rat CD2 domain 1 has been determined by NMR spectroscopy and X-ray crystallography, confirming the predictions from the sequence. Applications of this method of insertion of disulphide residues for probing protein structures are discussed, together with other structures of IgSF domains lacking the typical inter-sheet disulphide bond.
Gray
F
F
MRC Cellular Immunology Research Unit, Sir William Dunn School of Pathology, University of Oxford, UK.
Cyster
J G
JG
Willis
A C
AC
Barclay
A N
AN
Williams
A F
AF
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D018801:CD2 Antigens; D016466:CHO Cells; D006224:Cricetinae; D004220:Disulfides / Q000276:immunology / Q000378:metabolism*; D000939:Epitopes / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D011971:Receptors, Immunologic / Q000235:genetics / Q000276:immunology*; D017422:Sequence Analysis, DNA; D012995:Solubility; D013601:T-Lymphocytes / Q000276:immunology*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D018801:CD2 Antigens; D004220:Disulfides; D000939:Epitopes; D011971:Receptors, Immunologic