Published online 27 May 2009 Nucleic Acids Research, 2009, Vol. 37, No. 14 4545–4558 doi:10.1093/nar/gkp445 The structure of CrgA from Neisseria meningitidis reveals a new octameric assembly state for LysR transcriptional regulators Sarah Sainsbury1, Laura A. Lane 2 , Jingshan Ren 1 , Robert J. Gilbert1, Nigel J. Saunders3, Carol V. Robinson 2 , David I. Stuart1 and Raymond J. Owens1,* 1The Oxford Protein Production Facility and Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK, 2The University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, UK and 3The Bacterial Pathogenesis and Functional Genomics Group, The Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE. UK Received December 5, 2008; Revised April 21, 2009; Accepted May 11, 2009 ABSTRACT LysR-type transcriptional regulators (LTTRs) form the largest family of bacterial regulators acting as both auto-repressors and activators of target promoters, controlling operons involved in a wide variety of cellular processes. The LTTR, CrgA, from the human pathogen Neisseria meningitidis, is upregulated during bacterial–host cell contact. Here, we report the crystal structures of both regu- latory domain and full-length CrgA, the first of a novel subclass of LTTRs that form octameric rings. Non-denaturing mass spectrometry analysis and analytical ultracentrifugation established that the octameric form of CrgA is the predominant species in solution in both the presence and absence of an oligonucleotide encompassing the CrgA-binding sequence. Furthermore, analysis of the isolated CrgA–DNA complex by mass spectrometry showed stabilization of a double octamer species upon DNA binding. Based on the observed structure and the mass spectrometry findings, a model is proposed in which a hexadecameric array of two CrgA oligo- mers binds to its DNA target site. INTRODUCTION The largest family of one-component DNA-binding tran- scription factors found in bacteria are the LysR-type tran- scriptional regulators (LTTRs). LTTRs regulate the expression of a wide variety of genes, including operons involved in amino acid metabolism, oxidative stress, deg- radation of aromatic compounds and bacterial virulence (1). They comprise an N-terminal DNA-binding domain (DBD) containing a winged helix turn-helix-motif (wHTH) joined by a long linker helix (LH), involved in oligomerization, to a C-terminal regulatory domain (1,2). The regulatory domains of LTTRs share a similar fold to periplasmic substrate-binding proteins (3) and constitute the sensor part of the molecule, binding eﬀector molecules (1,4) or responding directly to redox-active compounds through the thiol groups of cysteine residues (5). LTTRs are dual function regulators acting as both auto-repressors and activators of target promoters, frequently of genes co-located with the LTTRs in the chromosome (1). Typically, an LTTR binds to a 50–60-bp stretch of DNA that comprises the recognition-binding site (RBS), containing an LTTR consensus-binding motif (T-N11-A), and an activation-binding site (ABS), which is usually in close proximity to the –35 promoter element. Transcriptional activation of the target gene is commonly dependent on the interaction of the LTTR with an eﬀector (1). This is often a substrate or metabolite of the regulated gene, which functions as a classical allosteric regulator, though LTTRs appear to remain bound to their cognate DNA targets under both inducing and non-inducing conditions. Reports of diﬀerential DNase protection of the ABS (6–8) and promoter DNA relaxation in the presence of an eﬀector suggest that the LTTR undergoes a conformational change upon eﬀector binding (7,8), which leads to either a quantitative and/or qualitative change in DNA binding. The molecular details of these events remain unclear since structural characterization of full-length LTTRs has proved challenging, largely due to their poor solubility (9,10). In fact, the structure of only one full-length LTTR has been reported to date, that of CbnR from Ralstonia eutropha NH9, which regulates an operon involved in the degradation of chlorocatechols (2). The crystal structure shows that CbnR assembles, as a dimer of dimers, into a homo-tetramer, which corresponds to the oligomeric state that has been reported for a number of other LTTRs (8,9,11,12). In contrast to *To whom correspondence should be addressed. Tel: 44 (0) 1865 287748; Fax: 44 (0) 1865 287748; Email: ray@strubi.ox.ac.uk 2009 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.