The role of protein sequence and global conformation in DNA binding specificity of members of the Cro family

Abstract

The Cro family of bacteriophage DNA‐binding proteins demonstrates the substantial conformational changes that can occur in protein evolution while the primary sequence is significantly conserved. Xfaso 1 and Pfl 6 of the Cro family are α‐helical and mixed α‐helical/β‐sheet, respectively, despite sharing 40% sequence identity. Both proteins are transcription factors that use a helix‐turn‐helix (HTH) motif as their primary DNA recognition moiety. However, the natural consensus DNA sequences of the proteins are different at three positions in each seven base‐pair half site. Fluorescence anisotropy measurements showed that wild‐type (WT) Xfaso 1 and Pfl 6 bound their cognate sites with dissociation constants (KD) of 230 nM and 56 nM, respectively. WT Pfl 6 bound its noncognate site with KD = 1.99 μM and WT Xfaso 1 did not bind its noncognate site at all. To explore whether this change in binding specificity is due to differences in sequence in the HTH region or to global differences in their tertiary structures, mutations were introduced into the HTH region of both proteins in order to equalize the binding region sequence while retaining global structure. The binding affinities of these variant proteins against the consensus binding sites were tested and preliminary results suggest that mutation of the Xfaso 1 sequence toward Pfl 6 allows it to bind the Pfl 6 consensus site. Funded by NSF (MCB‐0647390) and HHMI (52005889).