Transitive homology-guided structural studies lead to discovery of Cro proteins with 40% sequence identity but different folds.

Proteins that share common ancestry may differ in structure and function because of divergent evolution of their amino acid sequences. For a typical diverse protein superfamily, the properties of a few scattered members are known from experiment. A satisfying picture of functional and structural evolution in relation to sequence changes, however, may require characterization of a larger, well chosen subset. Here, we employ a "stepping-stone" method, based on transitive homology, to target sequences intermediate between two related proteins with known divergent properties. We apply the approach to the question of how new protein folds can evolve from preexisting folds and, in particular, to an evolutionary change in secondary structure and oligomeric state in the Cro family of bacteriophage transcription factors, initially identified by sequence-structure comparison of distant homologs from phages P22 and lambda. We report crystal structures of two Cro proteins, Xfaso 1 and Pfl 6, with sequences intermediate between those of P22 and lambda. The domains show 40% sequence identity but differ by switching of alpha-helix to beta-sheet in a C-terminal region spanning approximately 25 residues. Sedimentation analysis also suggests a correlation between helix-to-sheet conversion and strengthened dimerization.