Crystal and solution studies reveal that the transcriptional regulator AcnR of Corynebacterium glutamicum is regulated by citrate-Mg2+ binding to a non-canonical pocket

Abstract

Corynebacterium glutamicum is an important industrial bacterium as well as a model organism for the order Corynebacteriales, whose citric acid cycle occupies a central position in energy and precursor supply. Expression of aconitase, which isomerizes citrate into isocitrate, is controlled by several transcriptional regulators, including the dimeric aconitase repressor AcnR, assigned by sequence identity to the TetR family. We report the structures of AcnR in two crystal forms together with ligand binding experiments and in vivo studies. First, there is a citrate-Mg(2+) moiety bound in both forms, not in the canonical TetR ligand binding site but rather in a second pocket more distant from the DNA binding domain. Second, the citrate-Mg(2+) binds with a KD of 6 mM, within the range of physiological significance. Third, citrate-Mg(2+) lowers the affinity of AcnR for its target DNA in vitro. Fourth, analyses of several AcnR point mutations provide evidence for the possible involvement of the corresponding residues in ligand binding, DNA binding, and signal transfer. AcnR derivatives defective in citrate-Mg(2+) binding severely inhibit growth of C. glutamicum on citrate. Finally, the structures do have a pocket corresponding to the canonical tetracycline site, and although we have not identified a ligand that binds there, comparison of the two crystal forms suggests differences in the region of the canonical pocket that may indicate a biological significance.

Keywords: Corynebacteriales; Crystal Structure; Gene Regulation; Ligand-binding Protein; Metabolic Regulation; Structural Biology; Transcription Regulation.

FIGURE 1.

Analysis of AcnR-DNA interaction by EMSAs. A, determination of the apparent K_D for the binding of AcnR to its operator in the acn promoter. A 2 nm concentration of a Cy3-labeled DNA fragment covering the promoter region of acn was incubated with increasing concentrations of AcnR and analyzed as described under “Experimental Procedures.” An apparent K_D of 7.7 ± 0.9 nm was determined for dimeric AcnR. B, competition experiment with specific and nonspecific DNA (unlabeled), 2 nm Cy3-labeled DNA of the acn promoter, and 25 nm AcnR dimer. Specific DNA was unlabeled acn promoter DNA (10 nm, 40 nm, 100 nm), and nonspecific DNA was a 468-bp fragment covering the promoter region of cg1848 (10, 40, and 100 nm).

FIGURE 2.

The AcnR fold. A, stereo view of the AcnR protomer color-ramped from red to dark blue. B, the AcnR dimer in Form I generated by the crystallographic 2-fold symmetry axis, colored by chain. In B, the citrate-Mg²⁺ is shown with a space-filling representation. Figs. 2–5 were created using CCP4mg (62).

FIGURE 3.

Sequence alignment of AcnR homologues. The conserved residues are highlighted in dark gray, whereas residues with similar physico-chemical properties are boxed in white. The residues hydrogen-bonding the citrate and magnesium ion are highlighted with a star.

FIGURE 4.

Electrostatic potential and residue conservation in the AcnR DNA binding region (A) and pockets (B and C). The protein is displayed as a surface colored by residue conservation (top) and electrostatic potential (bottom). Residue conservation ranges from fully conserved in purple to not conserved (cyan). Citrate (green carbons) is shown as cylinders in C.

FIGURE 5.

The citrate binding distal pocket. A, stereo view with the citrate and residues contacting the citrate or magnesium depicted as cylinders. The 2F_o − F_c map contoured at the 1σ level is shown for the citrate-Mg²⁺. B and C, two views of the citrate-Mg²⁺ bound in the pocket of AcnR with the protein surface displayed to emphasize the shape of the pocket. D, superposition of the heavy atom derivative and native Form I structures. The 2F_o − F_c map at 1σ is shown for the gold ion. The gold in the derivative crystal displaces the citrate-Mg²⁺.

FIGURE 6.

Different states of the AcnR repressor. Left, scheme of the different functional forms found in TetR repressor and those found in this work for AcnR. Right, comparison of the two independent AcnR protomers in the asymmetric unit (blue and coral) in crystal Form II. The two protomers are superposed, and the movements of α6 and the DBD relative to the LBD are evident.

FIGURE 7.

Surface plasmon resonance titration of citrate-Mg²⁺ over AcnR. Left, sensorgram showing responses at the different ligand concentrations. Right, 1:1 ratio Langmuir curve fitted to the resulting responses.

FIGURE 8.

Bandshifts showing the effect of citrate-Mg²⁺ on AcnR-DNA binding. A, influence of Mg²⁺ and citrate on AcnR-DNA binding, separately and in combination. A 2 nm concentration of a Cy3-labeled DNA fragment covering the promoter region of acn was incubated with AcnR, Mg²⁺, and/or citrate as indicated. Only if both citrate and Mg²⁺ are added can a backshift be observed. B, reversibility of the citrate-Mg²⁺ effect. AcnR was incubated with DNA in binding buffer. A sample was taken (2), and the remaining complex was supplemented with Mg²⁺ and citrate. After a further 5 min, another sample was taken (3). EDTA was added, and another sample was taken after 5 min (4). C, influence of citrate-Mg²⁺ on mutational variants of AcnR. Arg-130 is involved in citrate binding, and the R130A variant no longer reacts to citrate-Mg²⁺. The same amino acid exchange at another position (Arg-92 within the P pocket) does not change the behavior of AcnR in the presence of citrate-Mg²⁺.

FIGURE 9.

Growth of C. glutamicum ΔacnR harboring different AcnR variants on glucose (A) and citrate (B). After precultivation in brain-heart infusion broth and CGXII glucose medium, cells of C. glutamicum ΔacnR carrying pEKEx2 plasmids encoding the native or a mutational variant of AcnR were cultivated in CGXII minimal medium containing either 111 mm glucose (A) or 50 mm sodium citrate and 5 mm CaCl₂ (B) at 30 °C and 1200 rpm in a Biolector® system. Averages and S.D. values (error bars) of three biological replicates are shown.