Functional modes of the regulatory arm of AraC

Abstract

One of the two crystal structures of the arm-dimerization domain determined in the absence of arbinose fails to show the arm, whereas the other structure does show it. The two structures lead to different pictures for the regulatory behavior of the arms. Trypsin digestion, fluorescence anisotropy, and NMR experiments presented here were designed to resolve the issue and show that in arm-dimerization domain, the arms are structured, although differently, in the presence and absence of arabinose. The arms have also been shown to interact with the DNA binding domains of AraC by their requirement for the immobilization of the DNA binding domains that is necessary for DNA looping and repression. The binding of arabinose has been shown to release the DNA binding domains and looping ceases. The picture resulting from the new experiments and the crystal structures of the arm-dimerization domain is that in the absence of arabinose, the arm adopts one structure on the dimerization domain and that the DNA binding domain then binds to this complex. Upon binding arabinose, the arm restructures and as a result, no longer serves as a gasket between the DNA binding domain and dimerization domain. The DNA binding domain is then released, subject only to the constraints imposed by the flexible linker connecting it to dimerization domain, and the protein relocates on the DNA and activates transcription.

Fig. 1

AraC and its mechanism of regulation. Schematic of the arabinose operon and AraC in its repressing and inducing states. The dimeric AraC protein in the absence of arabinose binds to araI₁ and araO₂ in a repressing state in which expression of the araBAD genes is low. The DNA binding and dimerization domains of AraC are connected by an interdomain linker, and the N-terminal arm is required in order that the protein bind to DNA in the repressing state. Arabinose leads the protein bind to the adjacent araI₁ and araI₂ half-sites from which it, in combination with the CAP protein, stimulate transcription of the araBAD genes.

Fig. 2

Trypsin sensitivity of arm-dimerization domain in the presence and absence of arabinose. Samples were prepared, digested, and analyzed as described in Methods. The various bands on the gel were identified by MALDI-TOF mass spectrometry. The “WT” protein carries the mutations Q5C, N16R, and C66S and the constitutive mutant protein carries in addition, L10D. Carbonic anhydrase, MW 30,000, and lysozyme, MW 14,300, were included as molecular weight standards.

Fig. 3

Fluorescence anisotropy of FITC-labeled AraC Q5C, N17R, C66S arm-dimerization domain in response to arabinose, circles; and glucose, squares.

Fig. 4

Overlay of the ¹H-¹⁵N HSQC spectra of AraC arm-dimerization domain, residues 2–166 in the presence of arabinose (red) and in the absence of arabinose (black). The contour level threshold has been adjusted to show only the more intense peaks. The stars and labels indicate the crosspeak positions of the AraC arm residues in free arm under identical solvent conditions. No cross-peaks were observed in these positions in either spectrum. The intense peaks that occur in pairs in the ¹⁵N 110–113 ppm region of the spectrum result from side chain amides.

Fig. 5

¹H-¹⁵N HSQC spectra of the AraC Arm+22 residue linker+DBD. (A) Contour levels adjusted to reveal the intense, sharp peaks deriving from unstructured residues. The cross-peaks deriving from the backbone amides of the AraC Arm residues are labeled. (B) Contour levels set to display all the amides of the arm-DBD. The data were acquired on a Varian INOVA spectrometer operating at a ¹H frequency of 800 MHz and equipped with a cryogenic probe. 852 and 204 complex points were collected with sweep widths of 13333 and 3403 Hz in ¹H and ¹⁵N resulting in acquisition times of 64ms (t₂) and 60ms (t₁), respectively. The 15N carrier frequency set at 118 ppm. Four transients were averaged for each t₁ point with a recycle delay of 1.1 second.

Fig. 6

Structures of the arm and dimerization domain. (A)Tube representation of residues 7–23 of the arabinose-bound (green) and the arabinose-free state (red) structures of the dimerization domain of AraC. Residues 24–30 and 32–166 of the arabinose-bound dimerization domain, subunit A from PDB file 2ARC, were RMS overlaid with the same regions of the apo structure of the dimerization domain, subunit D, from PDB 1XJA and are shown in blue-gray. (B) Structure comparison of the N-terminal arm. Differences in the phi (open circles) and psi (filled squares) values for residues 7–25 between the arm structures in the plus arabinose (2ARC) and apo structure of the Y31V mutant (1XJA) for each position in the arm.