As described above, the evolution of a protein domain is generally the result of a combination of a series of random mutations and a selection constraint imposed on function, i.e., the interaction with a ligand. The interaction between protein and ligand can be imagined as disturbances of the protein's energy landscape, which in turn bring about specific, three-dimensional changes in the protein structure [44, 45] . Binding energies however, need not be smoothly distributed over the protein's binding pocket as a limited number of amino acids may account for most of the free-energy change that occurs upon binding [45] [46] [47] . In these cases, new binding specificities (including loss of binding) may therefore arise through mutations at these hot spots. An example is a recent study of the PDZ domain in which it was shown that only a selected set of residues, and in particular the first residue of -helix 2 ( B1), directly confers binding to a set of C-terminal peptides [48] .