Crystal Structure of the Caseinolytic Protease Gene Regulator, a Transcriptional Activator in Actinomycetes

摘要

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 Å, 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 ∼30 Å, 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. 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 Å, 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 ∼30 Å, 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. Proteolysis in bacterial cells is a key cellular function. ATP-dependent self-compartmentalizing proteases, whose active sites are located in an inner cavity, play a dominant role in this process. Of these proteases, the serine protease caseinolytic protease (Clp) 3The abbreviations used are: Clp, caseinolytic protease; ClgR, clp gene regulator; HTH, helix-turn-helix; MPD, 2-methyl-2,4-pentanediol; EMSA, electrophoretic mobility shift assay; r.m.s.d., root mean square deviation.3The abbreviations used are: Clp, caseinolytic protease; ClgR, clp gene regulator; HTH, helix-turn-helix; MPD, 2-methyl-2,4-pentanediol; EMSA, electrophoretic mobility shift assay; r.m.s.d., root mean square deviation. has been extensively studied both mechanistically and functionally. The Clp proteases are a crucial part of the cell's protein quality control system disposing of truncated, misfolded, aggregated, or denatured proteins. In addition, Clp performs regulatory functions controlling the abundance of certain regulatory proteins in the cell. The activity of a number of regulator proteins (e.g. ComK (1Turgay K. Hahn J. Burghoorn J. Dubnau D. EMBO J. 1998; 17: 6730-6738Crossref PubMed Google Scholar), SpoIIAB (2Pan Q. Garsin D.A. Losick R. Mol. Cell. 2001; 8: 873-883Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), Spx (3Nakano S. Zheng G. Nakano M.M. Zuber P. J. Bacteriol. 2002; 184: 3664-3670Crossref PubMed Scopus (86) Google Scholar), and CtsR (4Kruger E. Zuhlke D. Witt E. Ludwig H. Hecker M. EMBO J. 2001; 20: 852-863Crossref PubMed Scopus (115) Google Scholar) in Bacillus subtilis, PopR in Streptomyces lividans (5Viala J. Mazodier P. Mol. Microbiol. 2002; 44: 633-643Crossref PubMed Scopus (26) Google Scholar)) is controlled through conditional proteolysis by Clp proteases. The concentration of the Clp subunits in the cell must be strictly controlled to ensure the capability to respond adequately to stress conditions resulting in a