The C repressor of the P2 bacteriophage
The C-repressor of bacteriophage P2 (P2 C) is a DNA-binding 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 λ-CII (Jain et al. 2005), the ω-repressors (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 15N 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 be in a homodimeric state. The crystal structure indicated five rigid helices in the N-terminus and a β-turn in the C-terminus. Since P2 C is a homodimeric protein in the absence of DNA, its dimer interface in solution has been determined with aid from the crystal structure assuming no conformational changes during the crystallization process.
Methods and results
An E. coli strain BL21(DE3) containing plasmid pEE679 expressing P2 C was grown at 310 K in M9 minimal medium containing 13C labeled-glucose, 15N labeled-NH4Cl and ampicillin (100 mg/ml) for 6–8 h until an OD600 = 0.6 was reached. Protein expression was induced by addition of isopropyl β-d-thiogalactoside (IPTG) to a final concentration of 1 mM at 37 °C for 4 h. The cells were harvested by centrifugation for 20 min at 9,000g at 4 °C and resuspended in 10 mM sodium phosphate buffer, pH 7.0. Cells were lysed by freezing/thawing together with sonication and thereafter centrifuged at 31,000g for 15 min at 277 K. The supernatant was collected and filtered with a 0.45 μm filter before starting the purification process. The protein was purified using ÄKTA™ FPLC-system in three consecutive steps. First, the filtered sample was adjusted to pH 8.0 with 5 M NaOH and loaded on a weak anion exchange column (DEAE, GE Healthcare) that had been equilibrated with 10 mM sodium phosphate buffer, pH 7.0 (running buffer). P2 C elutes with the flow through, as the pH of the running buffer is lower than the pI of P2 C. The second step was affinity chromatography using a HiTrap Heparin HP column equilibrated with running buffer. P2 C was eluted by a nine-column volume gradient of 1 M NaCl. The eluted fractions contain P2 C were loaded on a Superdex 200 gel filtration column (GE Healthcare) for further purification using 10 mM Na-Phosphate buffer, pH 7.0, 150 mM NaCl as running buffer. Finally, the sample was concentrated to 6 mg/ml using Amicon Ultra-15 centrifugal tubes (Millipore) with molecular weight cutoff 5 kDa. D2O was added to a final concentration of 10 % before the protein was transferred into a 5 mm NMR tube.
Structural Statistics for P2 C
Average target function (A2)
2.9 ± 0.18
Upper distance limits
Short-range, |i − j| ≤ 1
Medium-range, 1 < |i − j| < 5
Long-range, |i − j| ≥ 5
Average RMSD to mean
0.34 ± 0.12
0.78 ± 0.1
Number of conformers
Restraints per residue
RMS deviation for bond lengths
RMS deviation for bond angles
Average of bad steric contacts/100 residues
Ramachandran quality (1–81)
Residues in most-favored regions
Residues in additionally allowed regions
Residues in generously allowed regions
Residues in disallowed regions
Backbone dynamics of P2 C
P2 C-DNA complex
Discussion and conclusions
We have determined the solution structure of the dimeric P2 C repressor protein. The solution structure of P2 C displays close agreement with the crystal structure with a backbone RMSD of 1.16 Å between the crystal structure and the NMR-structure with the lowest CYANA target function for residues 5–81. This is crucial for the structure calculations since the five manually assigned inter domain NOEs were inferred based on shorter inter atomic distances for inter domain pairs. If the crystal and solution structures were not similar those five assignments would potentially be incorrect. Incorrect inter domain constraints would guide the structure calculation towards an incorrect local minimum of the target function. In addition to the a priori argument for similarity between the crystal and solution structures, such as secondary chemical shifts, 15N-relaxation data and mutation studies, a large body of evidence accumulates during the structure calculation. In particular more than 4000 NOEs including more than 100 inter domain and the convergence to a tight ensemble of structures strikingly similar to the crystal structure support the initial assumption.
The final 20 lowest target function structures were evaluated using PROCHECK_NMR (Morris et al. 1992). All dihedral angles for structured residues are in allowed conformations of Ramachandran map (Lovell et al. 2003
The C-terminus is shown to be flexible in solution from NMR data with very low order parameters, random coil chemical shift index and the absence of NOESY peaks. In the crystal structure no electron density is observed for the C-terminal residues after G85, also indicating that the C-terminal is disordered.
The C-terminus appears flexible, also in the 15N relaxation analysis, while the well-folded part of the sequence (residues 4–81) appears rigid. For the flexible C-terminus (relaxation data from residues 87–98) the extended model free model (Clore et al. 1990), i.e. model 4, is preferred. The internal correlation times are several hundred picoseconds and generalized squared order parameters for the slow internal component, i.e. Ss2 are below 0.33 and the fast components, Sf2, are in the range of 0.71–0.81. For the well-folded part the F-test indicates that model 1 is preferred with S2 in the range 0.74–0.96. The turn connecting helices 2 and 3 displays lower S2, while other turns appear as rigid as the elements of secondary structure on the picoseconds to nanosecond timescale. For residues T4 and F5 in the N-terminus, the model-free model (Lipari and Szabo 1982), i.e. model 2, is preferred with order parameters of 0.83 or higher and internal correlation times in the tens of picoseconds regime. For four residues (N51, I52, F65 and M66) the F-test indicates that significant exchange broadening contributes to R2 (the exchange contributions to R2 are 11, 13, 20 and 16 s−1 for N51, I52, F65 and M66, respectively). It is noteworthy that the local structures of the residues displaying exchange broadening are somewhat different in the crystal and solution structures. XTLSSR (King and Johnson, 1999) identifies N51 and I52 as members of a 310-helix in 23 % of the members of the ensemble, while they are classified as α-helical in the other ensemble members. In the crystal structure (2xcj) XTLSSR classifies them as α-helical. Residues F65 and M66 are classified as hydrogen bonded turn in most ensemble members, while they are classified as α-helical in 18 % of the members. In the crystal structure they are classified as α-helical. It is tempting to hypothesis that the observed uncertainties in the local structure for those sites are genuine features of the protein as they are consistent with exchange broadening.
The observable HSQC-peaks in the complex with DNA demonstrate that the C-terminus remains flexible also in the presence of DNA. This may be somewhat surprising as the C-terminus displays significant sequence identity to C-proteins from related phages (Massad et al. 2010). However, the finding that the C-terminus is flexible also in the complex with DNA explain an in vitro activity assays which demonstrated that a C-terminally truncated variant P2 C (1–90) is capable of binding to the target DNA and function as a repressor of a reporter gene (Massad et al. 2010).
A few signals were tentatively assigned to residues part of the helix bundle (T55, L81 and A82). If correctly assigned, this would indicate a significantly increased flexibility of those residues upon binding to DNA. The absence of HSQC peaks from the rigid core of the protein is caused by rapid relaxation, likely caused by slow tumbling of the 66 kDa complex and hence provides some evidence in support of the tetrameric binding model. However, exchange between complexes with different stochiometry could lead to signal loss through exchange broadening, and cannot be completely ruled out based on the current data. Solving the crystal and solution structures of P2 C has opened way for many questions regarding how a small protein like P2 C can bind such long DNA stretch (the center-to-center distance between O1 and O2 half sites is 22 bp). In addition, having the C-terminus very flexible even upon binding the DNA raises questions regarding its biological role.
We acknowledge Professor Elisabeth Haggård-Ljungquist for the important discussions and experimental assistance in the P2 C overexpression and purification. Dr. Isabella Felli and the CERM Magnetic Resonance Center in Florence is gratefully acknowledged for kind assistance and use of their NMR equipment. Professor Göran Karlsson from the Swedish NMR centre is acknowledged for the assistance in the NOESY experiments. Carl Trygger and Magn. Bergvall foundations are acknowledged for financial support to P.D. This work was also supported by grants from the Swedish Research Council (2010-5200 and 2014-5667), the Swedish cancer society, the Carl Trygger, Magn. Bergvall and Wenner-Gren foundations and to P.S.
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