Abstract
Human pathogens of the genera Corynebacterium and Mycobacterium possess the transcriptional activator ClgR (clp gene regulator) which in Corynebacterium glutamicum has been shown to regulate the expression of the ClpCP protease genes. ClgR specifically binds to pseudo-palindromic operator regions upstream of clpC and clpP1P2. Here, we present the first crystal structure of a ClgR protein from C. glutamicum. The structure was determined from two different crystal forms to resolutions of 1.75 and 2.05 A, respectively. ClgR folds into a five-helix bundle with a helix-turn-helix motif typical for DNA-binding proteins. Upon dimerization the two DNA-recognition helices are arranged opposite to each other at the protein surface in a distance of approximately 30 A, which suggests that they bind into two adjacent major grooves of B-DNA in an anti-parallel manner. A binding pocket is situated at a strategic position in the dimer interface and could possess a regulatory role altering the positions of the DNA-binding helices.
Highlights
The concentration of the caseinolytic protease (Clp) subunits in the cell must be strictly controlled to ensure the capability to respond adequately to stress conditions resulting in accumulation of truncated or misfolded proteins
According to genome-based searches, ClgR homologs are present in most members of the Actinomycetales, an order of high GϩC Gram-positive bacteria that comprises prominent pathogenic species like Mycobacterium tuberculosis and Corynebacterium diphtheriae
It was shown that ClgR from M. tuberculosis is capable of replacing ClgR in C. glutamicum enhancing expression of the clpC and clpP1P2 genes as well [10]
Summary
Protein Expression and Purification—The cloning, expression, and purification of the ClgR construct used for crystallization was performed as described previously by Engels et al [10]. Crystals of selenomethionine-labeled ClgR were obtained after 5 days by mixing 2 l of protein with 1 l of reservoir solution (0.0085 M CoCl2, 0.85 M 1,6-hexanediol, 15% glycerol, 0.085 M sodium acetate, pH 4.6). The crystals with an average size of 60 ϫ 40 ϫ 40 m3 grew after 1 month from a solution containing 0.085 M HEPES, pH 7.5, 8.5% polyethylene glycol 8000, and 15% glycerol They belong to space group P43212 with cell constants of a ϭ b ϭ 55.1, c ϭ 129.6 Å. Phases to 2.7-Å resolution were determined from a multiwavelength anomalous dispersion experiment carried out on a selenomethionine-labeled crystal on beamline X10SA at the Swiss Light Source using a Mar225 detector (Marresearch) [18]. Final refinement was performed against the native data to 2.05-Å resolution without the use of non-crystallographic symmetry restraints. After incubation of the DNA/ClgR mixtures for 30 min at room temperature, the samples were separated on a 12% native polyacrylamide gel, stained with Sybr-Green I, and photographed
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