Key interactions in neocarzinostatin, a protein of the immunoglobulin fold family

Neocarzinostatin (NCS) is a seven-stranded β-sandwich protein, the folding of which is similar to that of the variable domains of immunoglobulins (Ig). The investigation of the backbone dynamics of apo-NCS [Izadi-Pruneyre et al. (2001) Protein Sci., 10, 2228–2240] enabled us to identify the involvement of long side-chain residues in maintaining the rigidity of this β-protein. In the perspective of using this protein for drug targeting, this raises the following question: do these residues also play a key role in the stabilization of the β-sheet? To investigate this problem, various genetically engineered variants were constructed by mutating these residues to amino acids with shorter aliphatic side chains. These substitutions have no effects on the global fold. However, an important destabilization of the protein, higher than that expected for a simple ‘large-to-small’ substitution of buried hydrophobic residues, is observed for three mutants, V34A, V21A and V95A. Interestingly, the nature of the residues in these positions is highly conserved in the other Ig-like proteins. The absence of an evolutionary relationship between NCS and the other Ig-like proteins strongly suggests that this hydrophobic core is characteristic of the Ig-fold itself.