Structural basis for the nuclease activity of a bacteriophage large terminase.

The DNA-packaging motor in tailed bacteriophages requires nuclease activity to ensure that the genome is packaged correctly. This nuclease activity is tightly regulated as the enzyme is inactive for the duration of DNA translocation. Here, we report the X-ray structure of the large terminase nuclease domain from bacteriophage SPP1. Similarity with the RNase H family endonucleases allowed interactions with the DNA to be predicted. A structure-based alignment with the distantly related T4 gp17 terminase shows the conservation of an extended beta-sheet and an auxiliary beta-hairpin that are not found in other RNase H family proteins. The model with DNA suggests that the beta-hairpin partly blocks the active site, and in vivo activity assays show that the nuclease domain is not functional in the absence of the ATPase domain. Here, we propose that the nuclease activity is regulated by movement of the beta-hairpin, altering active site access and the orientation of catalytically essential residues.

Structural investigation of transcriptional regulator HlyIIR: influence of a disordered region on protein fold and dimerization.

B. cereus HlyIIR belongs to the TetR family of dimeric transcriptional regulators. Unlike other members of the TetR family, HlyIIR contains an insert between alpha-helices alpha8 and alpha9, which is located at the subunit-subunit interface. N-terminal segment of this insert (amino acids, Pro161-Ser169) forms a short alpha-helix alpha8* that occupies a complementary cavity on the surface of the adjacent subunit, whereas the C-terminal segment comprising 16 amino acids (Leu170-Glu185) is disordered. To understand whether this disordered segment is important for protein's function, we determined crystal structures of two engineered HlyIIR proteins where this segment was either substituted by a seven-residue flexible Ser-Gly linker or replaced by a cleavable peptide containing proteolytic sites at both ends. Unexpectedly, alteration or proteolytic removal of the disordered segment resulted in changes in protein's conformation and in a remarkable rearrangement at the subunit-subunit interface. X-ray structures of the two engineered proteins revealed an unusual plasticity at the dimerization interface of HlyIIR enabling it to form dimers stabilized by different sets of interactions. Structural comparison indicates that in spite of the flexible nature of the disordered segment, it is critical for maintaining the native structure as it influences the position of alpha8*. The data demonstrate how disordered loops on protein surfaces may affect folding and subunit-subunit interactions.

Papillomavirus E1 helicase assembly maintains an asymmetric state in the absence of DNA and nucleotide cofactors.

Concerted, stochastic and sequential mechanisms of action have been proposed for different hexameric AAA+ molecular motors. Here we report the crystal structure of the E1 helicase from bovine papillomavirus, where asymmetric assembly is for the first time observed in the absence of nucleotide cofactors and DNA. Surprisingly, the ATP-binding sites adopt specific conformations linked to positional changes in the DNA-binding hairpins, which follow a wave-like trajectory, as observed previously in the E1/DNA/ADP complex. The protein's assembly thus maintains such an asymmetric state in the absence of DNA and nucleotide cofactors, allowing consideration of the E1 helicase action as the propagation of a conformational wave around the protein ring. The data imply that the wave's propagation within the AAA+ domains is not necessarily coupled with a strictly sequential hydrolysis of ATP. Since a single ATP hydrolysis event would affect the whole hexamer, such events may simply serve to rectify the direction of the wave's motion.

Crystal structure of Bacillus cereus HlyIIR, a transcriptional regulator of the gene for pore-forming toxin hemolysin II.

Production of Bacillus cereus and Bacillus anthracis toxins is controlled by a number of transcriptional regulators. Here we report the crystal structure of B. cereus HlyIIR, a regulator of the gene encoding the pore-forming toxin hemolysin II. We show that HlyIIR forms a tight dimer with a fold and overall architecture similar to the TetR family of repressors. A remarkable feature of the structure is a large internal cavity with a volume of 550 A(3) suggesting that the activity of HlyIIR is modulated by binding of a ligand, which triggers the toxin production. Virtual ligand library screening shows that this pocket can accommodate compounds with molecular masses of up to 400-500 Da. Based on structural data and previous biochemical evidence, we propose a model for HlyIIR interaction with the DNA.

Two HlyIIR dimers bind to a long perfect inverted repeat in the operator of the hemolysin II gene from Bacillus cereus.

HlyIIR is a negative transcriptional regulator of hemolysin II gene from B. cereus. It binds to a long DNA perfect inverted repeat (44bp) located upstream the hlyII gene. Here we show that HlyIIR is dimeric in solution and in bacterial cells. No protein-protein interactions between dimers and no significant modification of target DNA conformation upon complex formation were observed. Two HlyIIR dimers were found to bind to native operator independently with Kd level in the nanomolar range. The minimal HlyIIR binding site was identified as a half of the long DNA perfect inverted repeat.