Structural determination of the phage 186 repressor protein

Bacteriophage 186 is closely related to the non-inducible P2 family of temperate phages based on genome homology. Unlike other P2 family members, phage 186 is SOS-inducible (in response to DNA damaging signals) and requires a cII gene for efficient lysogeny, similar to the lambda (λ) family. 186 and λ have common features that serve the same role in both phages: ensuring stable lysogeny and allowing for efficient switching to lytic growth upon induction. The lysogenic state of 186, like λ, is maintained by a repressor protein, the product of the gene cI. However, the λ and 186 operons differ in promoter arrangement, DNA binding sites, and the SOS response. Since bacteriophage λ has been extensively studied, the differences between λ and 186 provide insight into gene regulation and an additional model for the “genetic switch”. In an effort to gain insight into the structural basis of cooperativity and to help elucidate the details of this genetic switch, we pursued the structural determination of the 186 repressor, cI. Biochemical studies indicate that 186 cI has two domains: an N-terminal DNA binding domain and a C-terminal oligomerization domain. The X-ray structure of the C-terminal domain was solved by multiwavelength anomalous diffraction (MAD) to 2.Å resolution. By introducing a cooperativity mutant identified by genetic screens to the full length construct of 186 cI, we were able to obtain crystals of the full length dimer (E146K). The X-ray structure of the full length dimer was solved by molecular replacement (MR) to 1.95 Å resolution. A comparison of the crystallographic structures indicate the overall fold of the C-terminal domain and helix-turn-helix (HTH) motif from 186 cI is remarkably similar to λ repressor. Although similar in their overall fold, the difference in sequence provides insight into how 186 cI works to maintain lysogeny. The structures not only deepen our understanding of the genetic studies, but also provide insight into the framework for understanding the mechanism by which repression occurs