4546 Nucleic Acids Research, 2009, Vol. 37, No. 14 full-length LTTRs, several regulatory domain structures of LTTRs have been described, including CysB (3), BenM, CatM (4) and Cbl (13). Examination of these structures has conﬁrmed earlier mutagenesis data (11,14,15) that the site of eﬀector interaction is located at the interface between the two subdomains of the C-terminal domain, termed RD-I and RD-II (4,5). Interaction with eﬀectors has been shown to be associated with a conformational change in the C-terminal domain though how this is transmitted to the DBD is not known (4,5). Thus, despite the central role of LTTRs in the regula- tion of numerous metabolic processes in bacteria, their mechanism of action remains poorly understood at a structural level. To address the relationship between LTTR structure and function, we have focused on con- tact-regulated gene A (CrgA), an LTTR from Neisseria meningitidis, which has been implicated in host–pathogen interactions. CrgA was initially identiﬁed as being induced upon contact of N. meningitidis with human epithelial cells (16) and appears to be part of a group of genes that are co-ordinately upregulated during initial adhesion (17). CrgA is an autorepressor and activates the expres- sion of the divergently orientated gene, modulator of drug activity gene (mdaB), a putative quinone NADPH oxidor- eductase, suggesting that it may be involved in the response of Neisseria to oxidative stress (18,19). Here, we report the crystal structure of CrgA representing the second full-length structure of an LTTR to be described. Unexpectedly, the structure of CrgA showed the assembly of the protein into an octameric ring-like structure, in marked contrast to the tetrameric form of CbnR, which has become accepted as the usual oligomeric state of LTTRs. Cluster analysis of LTTR sequences showed that CrgA was associated with a distinct subset of LTTRs and may therefore deﬁne a novel subclass of reg- ulators that function as octamers. Non-denaturing mass spectrometry (MS) and sedimentation velocity analytical ultracentrifugation (AUC) were used to analyze the stoi- chiometry and relative stability of apo CrgA and holo CrgA in complex with its DNA target. On the basis of the stoichiometry of CrgA:DNA complexes detected and the octameric crystal structure, we propose a model of how CrgA interacts with its DNA tar- get sequence and discuss the wider signiﬁcance of these ﬁndings for transcriptional regulation by the LTTR family. MATERIALS AND METHODS Crystallography and structure solution The crystallization of CrgA and data collection methods have been reported previously (20). The structure of the selenomethionine derivative of the regulatory domain (residues 89–300) of CrgA was determined by multiple- wavelength anomalous dispersion (MAD) methods. Crystals of RD–CrgA were grown from protein at 3.9 mg ml–1 in 20 mM Tris pH 7.5, 200 mM NaCl, 1mM TCEP, using an additive screen (Hampton Research) opti- mization procedure in 200 mM ammonium acetate, 25% (w/v) polyethylene glycol 3350, 0.1 M bis–Tris pH 5.5 (21). The SHELX program suite was used to solve the selenium substructure (22) from data collected from Crystal I. SOLVE/RESOLVE (23) were used for the reﬁnement of selenium positions and phase extension to a resolution of 2.3 A˚ using data collected from a second crystal. The model was built manually using COOT (24) and reﬁned with CNS (25) using simulated annealing and positional reﬁnement with main chain NCS restraints followed by individual isotropic B factor reﬁnement. The ﬁnal stages of reﬁnement were carried out with REFMAC (26) from the CCP4 Suite (27) using TLS followed by restrained reﬁnement. The structure validation programs PROCHECK (28) and RAMPAGE (29) were used to assess the reﬁned structures. The ﬁnal reﬁned model has an R factor of 20.1% (Rfree 25.8%) with all residues in the allowed regions and 99.2% of residues in the favored region of the Ramachandran plot. The FL–CrgA protein was concentrated to 8.2 mg–1 in 20 mM citrate, 200 mM NaCl supplemented with 200 mM NDSB-256 (Hampton). Crystals of form A were grown in 10% (v/v) 2-methyl-2-4-pentanediol in 0.1 M sodium acetate pH 5.0. Crystals of form B were grown in 0.4 M ammonium sulfate, 0.8 M lithium sulfate and 0.08 M tri- sodium citrate pH 5.6. The full-length structure of CrgA (Crystal form A) was determined to a resolution of 3.2 A˚ by molecular replacement using MOLREP (30). A native Patterson and MOLREP indicated the presence of a pseudo-translation. Using the reﬁned regulatory domain of CrgA as the model an initial solution containing six molecules was found. Residues 1–87 from the CbnR struc- ture (PDB code 1IZ1) was used in a further molecular replacement experiment to locate four DBD pairs of CrgA. The two remaining regulatory domains were placed manually using the position of the DBD pairs as a guide. Each CrgA monomer was split into three NCS restraint groups, DBD, LH and regulatory domain. Density modiﬁcation and averaging was carried out with GAP (Jonathan M. Grimes and David I Stuart, unpub- lished program). The model was reﬁned using rigid body reﬁnement and then subjected to iterative rounds of manual rebuilding using COOT and TLS and restrained reﬁnement in REFMAC. The ﬁnal model has an R factor of 21.8% (Rfree 28.6%) with >95% of the residues in the favored region and 0.3% in the disallowed regions of the Ramachandran plot (29). The structure was determined for crystal form B using MOLREP with a dimeric unit of Crystal form A as the model. After rigid body reﬁnement with REFMAC the R factor was 29.3% (Rfree 40.0%) for data between 3.8 and 30 A˚ . No further reﬁnement was carried out due to the lower resolution. The data collection and reﬁnement statistics are presented in Tables 1 and 2. Representative electron density for the reﬁned models of CrgA, are shown in Supplementary Figure S5. The atomic coordinates of the regulatory domain and full-length CrgA structures have been deposited in the Protein Data Bank under the accession codes 3HHF and 3HHG, respectively.