Solution structure of the DNA binding domain of AraC protein

Abstract

We report the solution structure of the DNA binding domain of the Escherichia coli regulatory protein AraC determined in the absence of DNA. The 20 lowest energy structures, determined on the basis of 1507 unambiguous nuclear Overhauser restraints and 180 angle restraints, are well resolved with a pair wise backbone root mean square deviation of 0.7 A. The protein, free of DNA, is well folded in solution and contains seven helices arranged in two semi-independent sub domains, each containing one helix-turn-helix DNA binding motif, joined by a 19 residue central helix. This solution structure is discussed in the context of extensive biochemical and physiological data on AraC and with respect to the DNA-bound structures of the MarA and Rob homologs.

Fig. 1

Schematic diagram of the AraC protein and the Ara operon. In the minus arabinose repressing state, left, the AraC DNA binding domains are held in a position that favors looping by binding to the two widely separated DNA half-sites araO₂ and araI₁. Upon binding arabinose, right, the arms reposition on the dimerization domains over the arabinose. This frees the DNA binding domains to reorient and allows binding to the adjacent direct repeat DNA half-sites araI₁ and araI₂ partially overlapping the RNA polymerase binding site p_BAD.

Fig. 2

Structure of AraC DBD. (a) Overlay of the CA traces of the ensemble of 20 structures with the lowest final target function values. The RMSD to the mean was 0.68 Å.(b) Representation of the lowest energy structure refined in explicit water. The orientation is such that the two DNA contacting helices, 3 and 6, are foremost. The two H-T-H motifs are light colored. The minimal contact region between the two helix-turn-helix subdomains is at the top center of the diagram.

Fig. 3

Overlays of subdomains of AraC DBD. (a) Ensemble of the 20 structures from Fig. 2b in which residues 4–57 were used for RMS positioning. The RMSD to the mean was 0.48 Å. (b) Like (a) but residues 57–103 were used. The RMSD to the mean was 0.49 Å.

Fig. 4

Alignment of AraC with homologs MarA and Rob. The upper line shows the sequence number corresponding to the AraC DBD used in this paper. Residue 1 corresponds to residue 175 in the native AraC. Alignment between the AraC-DBD sequence, the complete MarA sequence and the Rob DNA binding domain is presented. Identical residues in all three are highlighted in black. Gray highlighted residues are similar or identical between AraC-DBD and either or both MarA and Rob. The boxes at the bottom correspond to alpha helical regions in AraC-DBD. The helix ends in the MarA and Rob structures are at identical positions +/− 1 residue of the indicated positions. The N-terminal and C-terminal H-T-H motifs comprise helices 2–3 and 5–6, respectively. The lowercase residues in AraC-DBD are the residues that were truncated prior to structure determination.

Fig. 5

Overlay of AraC and MarA. The solution structure of AraC-DBD, dark, overlaid with the DNA-bound structure of MarA, light. Only backbone atoms of the C-terminal H-T-H regions were used to calculate the overlay, which had an RMS difference of 1.3 Å.