1. Introduction
The regulatory protein AraC of the L-arabinose operon inEscherichia coli is a homodimer, where each subunit contains two domains: a dimerizing domain (DD) that binds arabinose (residues 1-167) and a DNA binding domain (DBD) (residues 175-281). The dimerized structure of the DD with (PDB entry 2ARC)1 and without (PDB entry 1XJA)2 bound arabinose has been determined by X-ray crystallography, and the DBD structure (PDB entry 2K9S) has been determined by NMR spectroscopy.3 The DD and DBD are connected by a 7-residue interdomain linker (residues 168-174). The structure of full length AraC in the presence or absence of arabinose, when bound to DNA, is not known.
In vivo, under inducing conditions, that is, in the presence of arabinose, one DBD binds to the araI1 half-site, and the other DBD binds to the nearby araI2 with four uncontacted bases (spacer ) between the two.4 Each DBD possesses two helix-turn-helix (HTH) motif regions that have been shown to contact DNA in two adjacent major grooves. When AraC is in the repressing state, it binds to the araI1 half-site and the araO2 half-site that is located 210 base pairs away to form a DNA loop. Upon the appearance of arabinose, the DBD that was bound to araO2 shifts and binds to araI2 .5 This flexibility in binding is facilitated by the ability of the DBDs to assume a variety of orientations and positions with respect to one another.6 Based on migration rates, DNA binding gel electrophoresis experiments indirectly indicated that in the presence of arabinose, AraC bends DNA by about 90° when the DBDs are bound toaraI1 (I1 ) and araI2(I2 ).7
The possible positioning of the DBDs when AraC binds toI1-spacer-I2 are limited by the known domain structures of AraC, the length and possible conformations of the interdomain linker, residue-residue contacts and residue-base contacts determined from experiments.6, 8, 9, 10 The length and binding energy constraints require that the DNA be appreciably bent in order to allow the two DBDs to contact the two half-sites. In this work, we determined experimentally the amount by which AraC bends DNA when it binds, and the positioning of the DBDs to gain an understanding of the structure of DNA-bound AraC in the presence of arabinose.
Atomic force microscopy (AFM) possesses the nanometer lateral resolution that is required to measure DNA bending. Modern AFMs can examine static samples in air (sample on mica), in vacuum, under water, at low temperatures, and can measure dynamic processes at video-rate speeds.11 AFM studies are feasible in air because during deposition onto divalent ion-treated mica substrate, DNA is able to equilibrate in two dimensions before adhering to the substrate and removal of the liquid buffer. Under such treatment, DNA is seen to resemble a worm-like chain with a persistence length of 53 nm on mica.12 Examples of AFM studies in air include the DNA bend angle induced by the binding of protein MutS on DNA mismatches,13 binding of Cro dimer protein to three operator sites,14 binding of Oct-1 as a monomer to the major groove of DNA,15 and binding of the drug cisplatin to DNA.16 In this paper, using static AFM measurements in air, we show that binding by AraC bends DNA by 69° ± 25° (one standard deviation).
We also modeled AraC binding to I1-spacer-I2 . Docking software was used to position each DBD onto DNA. Because no docking software is capable of handling the full flexibility of DNA,17 we presented the software with multiple DNA conformations and chose those that yielded the best surface complementarity after computer docking. Docking software also positioned one of the DBDs onto the DD with the limitation that protein side chain flexibility was not incorporated in the process. Our goal was to determine the tertiary and quaternary structure of AraC bound to DNA. Details on hydrogen bonding and molecular dynamics to handle residue and base pair flexibility were not included in this study. Satisfactory positioning of both DBDs required a bend of the I1-I2 binding site in the range of 52° - 88°.