NMR resonance assignments of NarE, a putative ADP-ribosylating toxin from Neisseria meningitidis 37 Expression of uniformly labeled 15N and 15N/13C proteins was carried out by growing BL21 strains in ISOGRO-15N and ISOGRO-15N, 13C rich media (Sigma), respectively. After buffer exchange and concentration, the resulting NMR samples contained approximately 0.6 mM NarE protein in 25 mM phosphate buffer (pH 7.5), 75 mM NaCl, 1 mM PMSF, a protease inhibitor cocktail (Boehringer), 1 mM NaN3, and either 90% H2O/10% D2O or 100% D2O. Trimethylsilyl-[2,2,3,3-2H4] propionate (TSP) was added as an internal 1H chemical shift refer- ence. 13C and 15N chemical shifts were referenced indi- rectly to TSP, using the absolute frequency ratios (Wishart et al. 1995). NMR experiments were performed at 27 C on a Bruker Avance-II 750 MHz equipped with a triple-resonance (1H, 15N, 13C) probe. Spectra recorded for sequential backbone assignment were as follows: 2D 15N–1H HSQC, 3D HNCO, 3D HN(CA)CO, 3D HNCA, 3D CBCA(CO)NH, 3D CBCANH, and 3D HBHA (CO)NH. Aliphatic and aromatic 1H and 13C side chain assignments were obtained using 3D HCCH-TOCSY, 3D HCCH-COSY, and 3D (H)CC(CO)NH experiments. All NMR data were processed with the NMRPipe software (Delaglio et al. 1995) and analysed with Sparky (Goddard and Kneller). Extent of assignments and data deposition Figure 1 shows the assigned 1H–15N HSQC spectrum recorded on the 15N-labeled protein sample. In this spec- trum most of the cross-peaks are well-dispersed, indicating that recombinant NarE is well-folded in the NMR aqueous buffer. All the NH backbone resonances were assigned except for the N-terminal residue M1 and for S49 for which the absence of a cross-peak may be due to loop mobility or conformational exchange as this amino acid follows a proline in the NarE sequence. The lack of 13C assignments for residue P48 due to line broadening con- ﬁrms the presence of dynamics in this region of the pro- tein. Interestingly, cysteines C67 and C128, which could be shown to be crucial for the coordination of iron (Del Vecchio et al. 2009), have a Cb chemical shift value higher than 40 ppm, suggesting the formation of an intramolec- ular disulﬁde bridge between them in the absence of a reducing agent. Since the used protein samples were col- ourless and line broadening of NMR signals due to para- magnetic effects of iron-III could not be observed, the presented resonance assignment likely refers to the protein’s apo-form. Overall, all residues corresponding to NarE were iden- tiﬁed: the backbone assignment is complete for 1Ha and reaches 98% for 1HN, 15N, 13Ca and 13CO nuclei. 88% of the aliphatic side chain resonances were assigned and 94% Fig. 2 Secondary structure prediction for the NarE sequence as obtained from TALOS? and consensus chemical shift index (CSI) programs. The regions identiﬁed to adopt a-helical and b-strand secondary structures are named a and b, respectively of the aromatic 1H and 13C resonances were identiﬁed including all Phe and Tyr residues. The assignment of NH2 side-chain resonances from asparagines and glutamines were also complete except for N145. The chemical shifts have been deposited into the BioMagResBank (http:// www.bmrb.wisc.edu) under accession number 16737. The CSI (Wishart and Sykes 1994) and TALOS? (Shen et al. 2009) programs were used to evaluate NarE secondary structure from 1Ha, 13Ca, 13CO and 13Cb chemical shifts. As shown in Fig. 2, both methods are in agreement with the presence of two a-helices (residues 11–15, and 76–81) and six b-strands (residues 4–9, 68–72, 88–96, 105–111, 118–125 and 136–143). Acknowledgments This work was supported by the Sixth Research Framework Programme of the European Community, FP6-STREP project ‘‘BacAbs’’, grant number LSHB-CT-2006-037325. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which per- mits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. References Althaus FR, Richter C (1987) ADP-ribosylation of proteins. Enzy- mology and biological signiﬁcance. Mol Biol Biochem Biophys 37:1–237 Del Vecchio M, Pogni R, Baratto MC, Nobbs A, Rappuoli R, Pizza M, Balducci E (2009) Identiﬁcation of an iron–sulfur cluster that modulates the enzymatic activity in NarE, a Neisseria menin- gitides ADP-ribosyltransferase. J Biol Chem 284(48):33040– 33047 Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293 Genco C, Wetzler L (eds) (2010) Neisseria: molecular mechanisms of pathogenesis. Caister Academic Press, Norwich, UK 123