Structural analysis of Ss-LrpB and Ss-LrpB:DNA complexes

Ss-LrpB is a transcriptional regulator from Sulfolobus solfataricus that belongs to the archaeal/bacterial Lrp family (Lrp=Leucine-responsive Regulatory Protein). Lrp-like proteins are composed of an N-terminal DNA-binding domain with a helix-turn-helix motif and a C-terminal alpha-beta-sandwich, also called RAM domain (RAM=Regulation of Amino acid Metabolism). These two domains are connected by a flexible linker. Due to the low solubility of Ss-LrpB, which behaves as a dimer in solution, the C-terminal domain was cloned separately as a His-tag derivative and purified, aiming at a structural analysis. This truncated protein comprises residues 69 to 147 of the full-length protein, omitting the hydrophobic linker. We succeeded in crystallizing this protein using the hanging-drop vapour-diffusion method in two different conditions (1). These crystals diffracted to 1.8 Å resolution. Preliminary attempts at molecular replacement with the C-terminal part of LrpA as model (27.8 % amino acid identity, 55.7 % similarity), as well the dimer as the monomer, have not been successful. In order to solve the structure a Se-Met derivative is being prepared. Ss-LrpB binds its own control region at three binding sites, called Box1, Box2 and Box3 (2). Atomic Force Microscopy (AFM) is becoming a widespread and very valuable technique in the study of protein-DNA interactions, resulting in a low resolution structural analysis of protein-DNA complexes. AFM allowed us to visualize the Ss-LrpB-DNA complexes, using a DNA fragment with the operator approximately in the centre (3). Complexes were formed at high Ss-LrpB concentration, in order to favorise full occupation of the operator binding sites. The vast majority of the nucleoprotein complexes shows two DNA arms extending from the complexed region, which has a globular shape. The contour length of 350 unbound DNA molecules and 350 complexes was measured and compared: this revealed a condensation of about 100 bp, which indicates the existence of very strong DNA deformations and even DNA wrapping. Bending angles were analysed using a method based on end-to-end-distances of the complexes. Altogether, these images strongly suggest wrapping of the operator around the regulator with an average bending angle of 260°. AFM images were also made using a Box2-mutant operator fragment, in which Box2 binding was weakened, allowing the observation of complexes bound at one or two Boxes. These observations lead us to the following hypothesis: at low Ss-LrpB concentrations, when only one Box is bound, a positive autoregulation might occur. At higher concentrations, when all three Boxes are bound, severe alterations in DNA conformation, caused by the DNA wrapping, would result in a negative autoregulation. Cooperativity is a major player in this interaction: small changes in Ss-LrpB concentration could therefore result in large changes in binding site occupancy and, accordingly, DNA conformation. This can lead to a fine-tuned switch between positive and negative regulation. We are also performing a more quantitative analysis of binding and cooperativity of the Ss-LrpB-operator interaction by studying binding to an elaborate series of operator mutants having deletions and/or substitutions in the linker regions connecting the three Boxes. This allows us to determine the importance of the relative orientation of the three Boxes and the importance of the exact length and sequence composition of the linkers.


References

1 Peeters, E., Hoa B.T.M., Zegers, I., Charlier, D. & Maes, D. (2005). Acta Cryst., F61, 985-988.
2. Peeters, E., Thia-Toong, T.-L., Gigot, D., Maes, D. and Charlier, D. (2004). Mol. Microbiol. 54: 321-336.
3. Peeters, E., Willaert, R., Maes, D. and Charlier, D. (2006). J. Biol. Chem. 281: 11721-11728.