The C repressor of the P2 bacteriophage

The C-repressor of bacteriophage P2 (P2 C) is a DNAbinding protein that controls the lifecycle of the P2 bacteriophage by directing it toward the lysogenic mode. P2 C is a 99 amino acids protein, which forms stable homodimers but not higher oligomers in the absence of DNA (Ahlgren-Berg et al. 2007). As opposed to the more common situation where dimeric proteins bind palindromic DNA-sequences, P2 C binds cooperatively to two direct repeats of DNA (named O1 and O2) flanking the -10 region in the Pe promoter in the genetic switch of the bacteriophage P2. The two 8 basepairs long direct repeats have a centre-to-centre distance of 22 base pairs (Ljungquist et al. 1984). According to a Electrophoretic Mobility Shift Assay (EMSA) analysis (Ahlgren-Berg et al. 2007), P2 C induces a high degree of bending of DNA upon binding. The puzzling question how a symmetric protein dimer can bind to an asymmetric DNA binding site where the epitope is repeated twice, as opposed to the more common inverted repeats. To the best of the authors’ knowledge, there are only three other examples of proteins that bind direct repeated DNA sequences in the protein databank. Those are the k-CII (Jain et al. 2005), the xrepressors (Weihofen et al. 2006) and the mammalian HOT1 (Kappei et al. 2013), which are all structural different to P2C (Massad et al. 2010). The DNA-binding epitope of P2 C is located in the N-terminus (residues 1–54), which contains a helix-turn-helix (HTH) motif (Eriksson et al. 2000; Massad et al. 2010). It has been reported that upon the superinfection of the satellite bacteriophage P4 of a P2 lysogenic cell, P4 is able to derepress the P2 lysogen (Liu and Haggård-Ljungquist 1999). This is mediated by binding the P4 E antirepressor to the P2 C after infection leading to the formation of multimeric complexes, thereby preventing the P2 C from binding to its operator (Liu and Haggård-Ljungquist 1999). Several mutations have been done on P2 C combined with activity assays to study the C-termini, the dimerization interface and the HTH motif, and to study the deactivation of P2 C by the P4 E antirepressor (Eriksson et al. 2000; Massad et al. 2010). One of the most interesting mutations is the truncation mutation performed on the last 9 residues of the C-terminus, which proved that the P2 C is still active even after truncation, indicating that the C-terminus might not be directly involved in the interaction with DNA. Solving the 3D structure of P2 C improves our understanding of its function and it is the first step to determine its DNA-binding mode. The backbone assignment of the P2 C has been published and deposited in the Biological Magnetic Resonance Bank (BMRB) under accession code 15577 (Massad et al. 2008). Here we report the solution structure of the P2 C together with the order parameters calculated from N relaxation data using the model-free approach. We have previously reported the crystal structure (PDB 2XCJ) of P2 C at 1.8 Å (Massad et al. 2010), where P2 C was shown to & Peter Damberg [email protected]