Structure and Function of the Arginine Repressor-Operator Complex from Bacillus subtilis James A. Garnett, Ferenc Marincs, Simon Baumberg†, Peter G. Stockley and Simon E. V. Phillips⁎ Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK Received 17 December 2007; received in revised form 29 February 2008; accepted 3 March 2008 Available online 12 March 2008 In many bacteria, the concentration of L-arginine is controlled by a trans- criptional regulator, the arginine repressor. In Bacillus subtilis this transcrip- tion factor is called AhrC and has roles in both the repression and activation of the genes involved in arginine metabolism. It interacts with 18 bp ARG boxes in the promoters of arginine biosynthetic and catabolic operons. AhrC is a hexamer and each subunit has two domains. The C-terminal domains form the core, mediating inter-subunit interactions and L-arginine binding, while the N-terminal domains contain a winged helix-turn-helix DNA- binding motif and are arranged around the periphery. Upon binding of the co-repressor L-arginine there is a ∼ 15° relative rotation between core C- terminal trimers. Here, we report the X-ray crystal structure of a dimer of the N-terminal domains of AhrC (NAhrC) in complex with an 18 bp DNA ARG box operator, refined to 2.85 Å resolution. Comparison of the N-terminal domains within this complex with those of the free domain reveals that the flexible β-wings of the DNA-binding motif in the free domain form a stable dimer interface in the protein–DNA complex, favouring correct orientation of the recognition helices. These are then positioned to insert into adjacent turns of the major groove of the ARG box, whilst the wings contact the minor groove. There are extensive contacts between the protein and the DNA phosphodiester backbone, as well as a number of direct hydrogen bonds between conserved amino acid side chains and bases. Combining this struc- ture with other crystal structures of other AhrC components, we have cons- tructed a model of the repression complex of AhrC at the B. subtilis bio- synthetic argC operator and, along with transcriptome data, analysed the origins of sequence specificity and arginine activation. © 2008 Elsevier Ltd. All rights reserved. Edited by K. Morikawa Keywords: arginine repressor/activator; crystal structure; structure-func- tion; gene array; transcriptional control Introduction The genetic control of arginine-metabolising en- zymes in many bacteria is achieved through the ar- ginine repressor, 1–4 which responds to intracellular levels of L-arginine. In Escherichia coli, where the ar- ginine repressor (ArgR) was first identified, the pro- tein controls a regulon of mostly separate genes involved in arginine biosynthesis. 3,5 It is also an essential component in the protein–DNA complex that resolves plasmid multimers during their repli- cation. 6 Bioinformatic analysis has revealed putative homologues of ArgR in many species. 7–9 The L-arginine analogue, L-arginine hydroxamate, is an antagonist of L-arginine, and Bacillus subtilis mutants resistant to it overproduce L-arginine. 10 Har- *Corresponding author. E-mail address: s.e.v.phillips@leeds.ac.uk. Present addresses: J. A. Garnett, Division of Molecular Biosciences, Imperial College London, South Kensington, London SW7 2AZ, UK; F. Marincs, Agricultural Biotechnology Center, Microarray Laboratory, Gödöllö, Hungary. † Deceased 11th April 2007. Abbreviations used: ArgR, arginine repressor; AhrC, arginine hydroxamate resistant mutant C; NCS, non-crystallographic symmetry. doi:10.1016/j.jmb.2008.03.007 J. Mol. Biol. (2008) 379, 284–298 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.