Crystal Structure of a Full-length LysR-type Transcriptional Regulator, CbnR: Unusual Combination of Two Subunit Forms and Molecular Bases for Causing and Changing DNA Bend

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Abstract

The LysR-type transcriptional regulator (LTTR) proteins are one of the most common transcriptional regulators in prokaryotes. Here we report the crystal structure of CbnR, which is one of the LTTRs derived from Ralstonia eutropha NH9. This is the first crystal structure of a full-length LTTR. CbnR was found to form a homo-tetramer, which seems to be a biologically active form. Surprisingly, the tetramer can be regarded as a dimer of dimers, whereby each dimer is composed of two subunits in different conformations. In the CbnR tetramer, the DNA-binding domains are located at the V-shaped bottom of the main body of the tetramer, and seem to be suitable to interact with a long stretch of the promoter DNA, which is approximately 60 bp. Interaction between the four DNA-binding domains and the two binding sites on the target DNA is likely to bend the target DNA along the V-shaped bottom of the CbnR tetramer. The relaxation of the bent DNA, which occurs upon inducer binding to CbnR, seems to be associated with a quaternary structure change of the tetramer.

Introduction

LysR-type transcriptional regulator (LTTR) proteins constitute one of the most common regulator families in prokaryotes.1., 2. To date, numerous LTTRs have been reported. Most of the LTTRs have a dual regulatory function, acting as transcriptional activators at one or several loci, while negatively regulating transcription of their own gene. The genes controlled by the LTTRs have diverse roles: amino acid biosynthesis, CO2 fixation, antibiotic resistance, catabolism of aromatic compounds, nodule formation of N2 fixing bacteria, synthesis of virulence factors etc.1

The LTTR proteins share amino acid sequence similarities over approximately 280 residues, with the strongest conservation of sequence in the N-terminal 65 residues. The N-terminal part of the LTTR proteins has been proposed to include a helix-turn-helix (HTH) motif that binds to a target DNA sequence. The C-terminal part of the proteins seems to be a regulatory domain, to which an inducer binds.1

To date, extensive efforts have been made to reveal the mechanism of transcriptional regulation by LTTR. These studies have revealed that (1) a typical LTTR binds to a long sequence of approximately 50–60 bp, which contains two distinct sites, a recognition-binding site (RBS) encompassing the inverted repeat motif including the T-N11-A sequence recognized primarily by LTTR, and an activation-binding site (ABS) which overlaps the −35 region of the transcriptional start site of the regulated gene; (2) the wide stretch of the DNA region containing RBS and ABS covered by LTTRs suggests that LTTRs bind to the DNA as a tetramer in its biologically active form when activating transcription of the regulated genes; (3) the DNA binding of LTTR causes a bend in the target DNA; and (4) the bent DNA is relaxed upon inducer binding to LTTR, leading to the formation of an active complex with RNA polymerase to initiate transcription.1., 3.

Tyrrell et al. have revealed the crystal structure of the regulatory domain of CysB, one of the LTTR proteins.4 The regulatory domain of CysB (hereafter CysB(RD)), which is composed of two sub-domains, is similar to that of the Lac repressor.5 The crystal structure and the analysis with its mutants have revealed that the inducer-binding site is located between the two sub-domains.6 It is intriguing to note that CysB(RD) forms a dimer in the crystal, although the CysB molecule forms a tetramer in solution.7 The lack of the DNA-binding domain seems to cause this difference. Although the crystal structure of the CysB(RD) has revealed the details of the inducer binding, details of the promoter recognition and the transcriptional activation mechanism upon the inducer binding remain unknown, due to lack of structural information on the entire molecule. To reveal these points, we have tried to solve the overall structure of LTTR proteins.

CbnR, a LTTR found in the 3-chlorobenzoate degradative bacterium Ralstonia eutropha NH9, activates the expression of the cbnABCD genes, which are responsible for the degradation of chlorocatechol converted from 3-chlorobenzoate and are transcribed divergently from cbnR.8 Biochemical analyses of CbnR have revealed that CbnR has typical characteristics of LTTRs. CbnR binds to the cbnA promoter region containing an inverted repeat motif and protects an approximately 60 bp region from DNase I digestion with the sites of hypersensitivity at nearly the center of the protected region. The CbnR binding to the promoter region induces the bending of the promoter DNA region. In the presence of the inducer molecule, cis,cis-muconate, the bending angle is relaxed from 78° to 54°.9 Since CbnR is a typical LTTR, understanding the crystal structure of CbnR will give insights into the general mechanism of the transcriptional regulation by LTTRs.

Here, we report the overall structure of CbnR. This is the first crystal structure of a full-length LTTR. The CbnR molecule forms a tetramer in the crystal, which seems to be a biologically active form. The tetramer is composed of two compact form subunits and two extended form subunits. The subunit is composed of two domains, the DNA-binding domain and the regulatory domain. All the DNA-binding domains are located at the bottom of the tetramer, and seem to be suitable to interact with a long stretch of the promoter DNA, which is approximately 60 bp.

Section snippets

Structure determination

CbnR was crystallized in three different forms, I, II, and III.10 Of the three, we have determined crystal structures of forms I and II. To determine the crystal structure by the multiple-wavelength anomalous diffraction (MAD) method, we have prepared selenomethionyl CbnR (Se-CbnR). The MAD phasing for the form I crystal was carried out at 2.79 Å resolution (Table 1). The MAD electron density map was applied to density modification, and the obtained map can be interpreted without ambiguity (

Structure of LTTR

The present crystal structure of CbnR is the first full-length structure of LTTR. It is likely that the structure of CbnR represents a common structure of LTTRs due to the amino acid sequence similarity among the LTTR family proteins (Figure 5). It has been proven that many of the LTTR proteins form tetramers in solution.7., 14., 15. The CbnR tetramer, thus, appears to represent a typical tetrameric structure of LTTR, which is likely to be composed of four identical subunits with two distinct

Crystallographic analysis

CbnR and Se-CbnR were overexpressed with E. coli and purified with two chromatographic steps as described.10 CbnR and Se-CbnR were crystallized by the hanging-drop, vapor-diffusion method, resulting in three different crystal forms.10 Of the three crystal forms, the crystal structures of forms I and II have been determined. The crystal structure of the CbnR in form I was determined by the MAD method. Analysis of the fluorescence from a form I crystal near the anomalous K-edge of selenium

Acknowledgements

We thank Dr K. Miura of JASRI for her assistance in the MAD data collection at BL40B2 of SPring-8. We also thank Drs Y. Kyogoku and Y. Fujiyoshi for encouragement. This study was partly supported by the Japan New Energy and Industrial Technology Development Organization (NEDO) Japan, Grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and a Grant-in-aid (Endocrine Disrupters) from the Ministry of Agriculture, Forestry, and Fisheries of Japan

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