1H, 13C and 15N assignment of the GNA1946 outer membrane lipoprotein from Neisseria meningitidis
- 835 Downloads
GNA1946 (Genome-derived Neisseria Antigen 1946) is a highly conserved exposed outer membrane lipoprotein from Neisseria meningitidis bacteria of 287 amino acid length (31 kDa). Although the structure of NMB1946 has been solved recently by X-Ray crystallography, understanding the behaviour of GNA1946 in aqueuos solution is highly relevant for the discovery of the antigenic determinants of the protein that will possibly lead to a more efficient vaccine development against virulent serogroup B strain of N.meningitidis. Here we report almost complete 1H, 13C and 15N resonance assignments of GNA1946 (residues 10–287) in aqueous buffer solution.
KeywordsPathogenic bacteria Meningitis Liporotein NMR Antigen
Neisseria meningitidis is an encapsulated, Gram-negative bacterium that colonizes the upper respiratory tract of humans. During invasive infection, the bacterium enters the bloodstream, where it multiplies to high density and causes a form of sepsis characterized by the dramatic disruption of the endothelium and microvasculature. From the bloodstream the bacterium can cross the blood–brain barrier and cause meningitis, which mostly affects infants, children, and adolescents who do not have bactericidal antibodies to the infecting strain. Although conjugate vaccines against serogroups A, C, Y, and W-135 were proven to be safe and effective in eliminating the disease, the poor immunogenicity of the serogroup B capsular polysaccharide and its cross-reactivity toward human tissues have stressed the need to develop a universal vaccine covering all meningococcal strains (Giuliani et al. 2006). A few years ago we determined the sequence of the genome of a meningococcus B strain (MC58) and used it to discover novel protective antigens (Pizza et al. 2000). Among them we identified GNA1946, a highly conserved lipoprotein sharing homology with periplasmic ABC methionine transporters (Pizza et al. 2000; Jacobsson et al. 2006; Peng et al. 2008). Yang and colleagues have recently solved the crystallographic structure of GNA1946 and postulated a high affinity binding to l-methionine, hypothesizing a role as an initial receptor of the ABC transporter family (Yang et al. 2009). In this study, we report the 1H, 13C and 15N chemical shift assignments of GNA1946 in aqueous solution.
Methods and experiments
Cloning, expression and purification of GNA1946 antigen
GNA1946 was amplified from a NMB genomic DNA library using the following primers NMB1946_BamHI_S; (ACTGGGATCCATGAAAACCTTCTTCAAAACCC) and NMB1946_XhoI_AS (ACTGCTCGAGTTATTTGGCTGCGCCTTCATTCC) and subsequently subcloned into pGEX-6P-1 vector using BamHI and XhoI as restriction sites. Uniformly 13C,15N-labelled protein was expressed in Escherichia coli C43(DE3) strain (Lucigen) in M9 medium containing 0.1% 15N-ammonium chloride, 0.2% 13C-glucose (Cambridge Isotope Laboratories) and 50 μg/ml ampicillin (Sigma). Protein expression was induced with 1 mM IPTG (isopropyl β-D-thiogalactosidase). After 16 h incubation at 293 K cells were centrifuged and the cell pellet was resuspended in GST buffer at pH 8.0 and lysed by sonication. The recombinant protein was purified using a column containing Glutathione Sepharose 4 Fast Flow (GE Healthcare) pre-equilibrated with GST buffer. Sample purity was monitored by SDS–PAGE. Thermolysin (Sigma) was used to remove the GST tag from the recombinant protein. After enzymatic reaction, the proteins were separated by gel filtration on a column packed with Superdex 200 (GE Healthcare). The protein-rich fractions were pooled and then subjected to a final purification step on Gluatahione Sepharose 4 Fast Flow column to remove the residual GST and uncleaved fused-protein. Finally GNA1946 was dialyzed three times against 100 volumes of 200 mM NaCl, 0.05% NaN3, 20 mM sodium phosphate buffer, pH 7.0, concentrated to 0.5 mM, supplied with 5% D2O and used directly for the NMR measurements.
All spectra were recorded at 298 K on a Bruker Avance 900 MHz spectrometer equipped with a cryoprobe. Sequence-specific resonance assignment was accomplished based on a combination of triple-resonance experiments as well as 15N- and 13C-HSQC-NOESY spectra. Backbone assignment was performed based on the set of HNCO/HN(CA)CO and HNCA/HN(CO)CA experiments (Yamazaki et al. 1994). Additionally, CBCANH and CBCA(CO)NH spectra (Shan et al. 1996) were evaluated whenever possible to confirm the assignments made and derive information on Cβ chemical shifts. Side-chain resonance assignments were accomplished with HCCH-TOCSY experiments (Kay et al. 1993). Finally, chemical shifts were obtained by picking peaks in a 13.3 ms constant-time 1H,13C-HSQC spectrum (Vuister and Bax 1992). The aromatic ring systems of Phe, Tyr, His, and Trp residues were picked in a 8.8 ms constant time 1H,13C-HSQC and correlated with β-carbons via the HBCBCGCDHD experiment (Yamazaki et al. 1993). All chemical shift values were finally derived from the position of peaks in the 1H,15N-HSQC and the constant time 1H,13C-HSQC spectra. All experiments employed pulsed-field gradients (Keeler et al. 1994). Data were processed with TOPSPIN 2.1 (Bruker) and analyzed with the CCPNMR Analysis 2.1 software (Vranken et al. 2005).
Assignment and data deposition
This work was supported by the Sixth Research Framework Programme of the European Community, FP6-STREP project ‘‘BacAbs’’, grant number LSHB-CT-2006-037325.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
- Shan X, Gardner KH, Muhandiram DR, Rao NS, Arrowsmith CH, Kay LE (1996) Assignment of N-15, C-13(alpha), C-13(beta), and HN resonances in an N-15, C-13, H-2 labeled 64 kDa trp repressor-operator complex using triple-resonance NMR spectroscopy and H-2-decoupling. J Am Chem Soc 118(28):6570–6579CrossRefGoogle Scholar
- Vuister GW, Bax A (1992) Resolution enhancement and spectral editing of uniformly 13C-enriched proteins by homonuclear broadband 13C decoupling. J Magn Reson 98:428–435Google Scholar