Phage P4 origin‐binding domain structure reveals a mechanism for regulation of DNA‐binding activity by homo‐ and heterodimerization of winged helix proteins

Summary The origin‐binding domain of the gpα protein of phage P4 (P4‐OBD) mediates origin recognition and regulation of gpα activity by the protein Cnr. We have determined the crystal structure of P4‐OBD at 2.95 Å resolution. The structure of P4‐OBD is that of a dimer with pseudo twofold symmetry. Each subunit has a winged helix topology with a unique structure among initiator proteins. The only structural homologue of the P4‐OBD subunit is the DNA‐binding domain of the eukaryotic transcriptional activator Rfx1. Based on this structural alignment, a model for origin recognition by the P4‐OBD dimer is suggested. P4‐OBD mutations that interfere with Cnr binding locate to the dimer interface, indicating that Cnr acts by disrupting the gpα dimer. P4‐OBD dimerization is mediated by helices α1 and α3 in both subunits, a mode of winged helix protein dimerization that is reminiscent of that of the eukaryotic transcription factors E2F and DP. This, in turn, suggests that Cnr is also a winged helix protein, a possibility that is supported by previously unreported sequence homologies between Cnr and Rfx1 and homology modelling. Hence, in a mechanism that appears to be conserved from phage to man, the DNA‐binding activity of winged helix proteins can be regulated by other winged helix proteins via the versatile use of the winged helix motif as a homo‐ or heterodimerization scaffold.