ONE SEQUENCE, TWO FOLDS: CRYSTAL STRUCTURE OF A MISFOLDED, METASTABLE FORM OF CD2. R. Leo Brady and Alison J. Murray, Department of Biochemistry, University of Bristol, Bristol BS8 1TD U.K.

We have recently determined the crystal structure of a misfolded, metastable form of the NH₂-terminal domain of the lymphocyte cell adhesion molecule CD2. When expressed as part of a glutathione-S-transferase fusion protein the polypeptide sequence for domain 1 of CD2 can fold in two different ways. The major (85%) monomeric component forms a familiar immunoglobulin superfamily fold as previously demonstrated by both X-ray crystallography and NMR spectroscopy. We now describe the structure of a second, dimeric form present in about 15% of recombinant CD2 molecules. After denaturation and refolding in the absence of the fusion partner dimeric CD2 is converted to monomer, illustrating the dimeric form represents a metastable folded state.

The crystal structure of this dimeric form, refined to 2.0 Å resolution, reveals two domains with overall similarity to the IgSF fold found in the monomer. However, in the dimer each domain is formed by the intercalation of two polypeptide chains. Hence each domain represents a distinct folding unit that can assemble in two different ways. In the dimer the two domains fold around a hydrophilic interface believed to mimic the cell adhesion interaction at the cell surface, and we demonstrate formation of dimer can be regulated by mutating single residues at this interface. This unusual misfolded form of the protein, which appears to result from inter- rather than intramolecular interactions being favoured by an intermediate structure formed during the folding process, illustrates that evolution of protein oligomers is possible from the sequence for a single protein domain. We suggest this exploitation of a finely-balanced folding intermediate capable of developing to two distinct folds may pre-date "domain swapping" as a mechanism for the evolution of protein oligomers. A biological role for the alternative fold cannot be ruled out, as in this state the adhesion interface is buried and effectively switched "off".

Analyses of a series of deletion and single-site mutations of CD2 support the proposed folding pathway for immunoglobulin superfamily domains implied by this structure. Identifying domain features important in directing folding pathways has important implications for protein design and engineering.