Crystal structure of the ligand-binding domain of a LysR-type transcriptional regulator: transcriptional activation via a rotary switch

Abstract

LysR-type transcriptional regulators (LTTRs) generally bind to target promoters in two conformations, depending on the availability of inducing ligands. OccR is an LTTR that regulates the octopine catabolism operon of Agrobacterium tumefaciens. OccR binds to a site located between the divergent occQ and occR promoters. Octopine triggers a conformational change that activates the occQ promoter, and does not affect autorepression. This change shortens the length of bound DNA and relaxes a high-angle DNA bend. Here, we describe the crystal structure of the ligand-binding domain (LBD) of OccR apoprotein and holoprotein. Pairs of LBDs form dimers with extensive hydrogen bonding, while pairs of dimers interact via a single helix, creating a tetramer interface. Octopine causes a 70° rotation of each dimer with respect to the opposite dimer, precisely at the tetramer interface. We modeled the DNA binding domain (DBD), linker helix and bound DNA onto the apoprotein and holoprotein. The two DBDs of the modeled apoprotein lie far apart and the bound DNA between them has a high-angle DNA bend. In contrast, the two DBDs of the holoprotein lie closer to each other, with a low DNA bend angle. This inter-dimer pivot fully explains earlier studies of this LTTR.

Figure 1.

Octopine regulated genes, PoccQ and PoccR promotors and proposed model for OccR regulation. A. OccR holoprotein activates a 13 gene operon that directs uptake and catabolism of octopine-type opines and includes traR, a quorum-sensing transcription factor. OccR also autorepresses. B. A portion of the intergenic region between occQ and occR, showing the PoccR and PoccQ promoter elements (underlined). The Recognition Sequences (RS1 and RS2) and Activation Sequence (AS) are also shown. Dyad repeats at RS1 are depicted in red. C. Previous biochemical experiments showed that the OccR apoprotein binds a 55 nucleotide region that includes RS1 and RS2, causing a sharp DNA bend and a nuclease-hypersensitive site at the center of the bound DNA. OccR holoprotein binds a 45 nucleotide region that includes RS1 and AS, and causes a low angle DNA bend. Only the holoprotein can activate the occQ operon, while both the holoprotein and apoprotein autorepress occR. Curved white arrows represent a putative pivot of one LBD dimer with respect to the other dimer.

Figure 2.

A. Ribbon diagram of one subunit of the OccR LBD apoprotein. Colors indicate primary sequence position, from N terminus (blue) to C terminus (red). Helices and strands are labelled according to the predicted structure of the full length protein (see Sup. Fig. 1A). B. Ribbon diagram of the corresponding holoprotein.

Figure 3.

A. Dimer and tetramer interfaces of the aproprotein tetramer. Two dimer interfaces are shown between strands β4 and β9, while a single tetramer interface is shown at helix α10. B. Half of the dimer interface, showing hydrogen bonds between subunits at strands β4 and β9 as well as between helices α6 and α11. Hydrogen bonds are found between Glu217 (backbone amine) and Val122 (backbone oxygen, 2.7 Å), between Thr223 (hydroxyl) and Ala102 (backbone oxygen, 3.5 Å), between Thr223 (hydroxyl) and Asn103 (hydroxyl, 2.4 Å), between Ser226 (hydroxyl) Asn103 (backbone oxygen, 3.0 Å), between Leu124 (backbone amine) and Glu217 (backbone oxygen, 2.6 Å) and between Gly126 (backbone amine) and Ser219 (hydroxyl, 2.8 Å). The same hydrogen bonds occur on the symmetric face of each dimer for a total of 12 hydrogen bonds in each dimer interface. C. The tetramer interface, showing a single water-mediated hydrogen bond (red), and numerous hydrophobic interactions (blue).

Figure 4.

The octopine binding site. A. Surface view of bound octopine. The pyruvyl moiety is exposed to solvent, while the arginyl group, and especially its gaunido group, is more deeply buried in the binding pocket. B. Electron density map of bound ligand. All ligand molecules have similar quality density (not shown). C. Hydrogen bonds between the two carboxyl groups of octopine and Ser129, Ser130, Ser221, His222, and the backbone of Ala100. D. Hydrogen bonds between the guanido group of octopine and Asp147, Asp238, and Asp261. These interactions help to account for the rather broad ligand specificity of OccR, as all octopine analogs with basic groups are strong inducers (Flores-Mireles et al., 2012).

Figure 5.

Conformational changes induced by octopine binding. A. Binding of octopine causes a large movement of a α10 and two adjacent loops (compare holoprotein in color with apoprotein in grey). This movement is necessary to avoid a steric clash between octopine and Thr195 and Thr197. B and C. Octopine causes a 70⁰ rotation between subunits B and C. The tetramer interface between subunits B and C is shown in the apoprotein (B) and the holoprotein (C). For clarity, the orientation of the B subunits are fixed in space while subunit C rotates in panel C relative to panel B.

Figure 6.

In the apoprotein, subunits A and B make extensive contacts with subunits C and D, while in the holoprotein, a deep cleft fully separates subunit A from subunit D, and the only contacts between subunits B and C occur at helix α10. In the full length protein, the tetramer would be further stabilized by the DBD and linker domains.

Figure 7.

A. Model of the OccR DNA binding domain (DBD), linker domain, and bound DNA, using the homologous regions of BenM as a template (Alanazi et al., 2013). B and C. Models of full-length apoprotein (B) and holoprotein (C). All four DBD and linker helices are depicted in green. For clarity, in comparing the two figures, the upper DNA fragment and subunits A and B (light blue and dark blue) were fixed in position to more clearly visualize the movement of the lower DNA fragment and the rotation of subunits C and D (orange and red). B. The apoprotein binds RS1 and RS2, protecting a 55 nucleotide region against DNase I (with a DNase hypersensitive site midway between RS1 and RS2), and causing a high angle DNA bend. OccR occupies the interior angle of this bend (Wang and Winans, 1995b). C. In the holoprotein, one dimer remains at RS1, while the other dimer shifts by 10 nucleotides from RS2 to AS, thereby shortening the binding site, abolishing the DNase I hypersensitive site, and decreasing the angle of the DNA bend. This model fully accounts for earlier genetic and biochemical studies of this protein (Akakura and Winans, 2002a, b; Tsai et al., 2011; Wang et al., 1992; Wang and Winans, 1995a, b).