Letter to the Editor: NMR assignments of Human RGS18
Victoria A. Higman1, Martina Leidert1, Annette Diehl1, Jonathan Elkins2, Meera Soundararajan2, Hartmut Oschkinat1 andLinda J. Ball2
1Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, D-13125 Berlin,Germany.
2Structural Genomics Consortium (SGC), University of Oxford, Botnar Research Centre, OxfordOX3 7LD,UK.
Corresponding author:
Dr. L. J. Ball
Structural Genomics Consortium (SGC), University of Oxford, Botnar Research Centre, OxfordOX3 7LD,UK.
Tel: +44 1865 227723; Fax: +44 1865 737231
E-mail:
Keywords: chemical shift, G-protein signalling, NMR, RGS domain
Abbreviations: RGS: regulator of G-protein signalling; NaPi: sodium phosphate buffer; IPTG: isopropyl-beta-D-thiogalactopyranoside
Character count: 7754Biological context
Regulators of G-protein signalling (RGS) proteins contain domains that terminate the signal emanating from G-protein coupled receptors (GPCRs). When a GPCR binds a specific ligand, a signal is generated that is transmitted to the G-proteins which consist of G and G subunits. In the inactive state, G and G exist as a complex with the G GTPase in the inactive GDP bound form. Upon activation, GDP is replaced with GTP which induces G and G complex dissociation. At this stage, both G and G proteins are active. G does have an intrinsic GTPase activity but this is greatly enhanced by the association with an RGS domain. Hence RGS proteins act as GTPase-activating proteins (GAPs). Once GTP hydrolysis has taken place, the GDP-Gprotein can recombine with G thus terminating the signal.
RGS18 is a member of the B/R4 subfamily of RGS proteins. All of these subfamily members have an N-terminal amphipathic helix which enhances the localisation of the protein to the membrane surface (Bernstein et al., 2000; Chen et al., 1999) where it is ideally placed to interact with the G-proteins. Its expression is most abundant in platelets and megakaryocytes and it binds to Gi1, Gi2, Gi3, and Gq but not Gz, Gs or G12 (Gagnon et al., 2002; Nagata et al., 2001). Although no direct link to any specific disease is yet proven, RGS18 is believed to play roles in the regulation of megakaryocyte differentiation, proliferation and chemotaxis (Yowe et al., 2001), in platelet activation (Gagnon et al., 2002), and as an inhibitor for chemokine-induced dendritic cell migration (Shi et al., 2004) acting primarily via modulation of Gq signalling (Park et al., 2001).
Methods and experiments
Cloning, expression and purification of human RGS18
The human RGS18 domain (residues 75-223 of RGS18_human, Q9NS28) was cloned into the homemade pLIC-SGC1 vector, which adds a TEV-cleavable hexahistidine tag to the N-terminus of the protein.Uniformly 15N- and 15N, 13C-labelled His-TEV-SM-RGS18 domains were grown in E. coli BL21(DE3)-Rosetta cells (Novagen) containing the above plasmid, in M9 minimal medium supplemented with 60 µg/ml carbenicillin, 30µg/ml chloramphenicol, and containing 0.5 g/l 15NH4Cl and either 2 g/l (w/v) 12C6-glucose or 13C6-glucose, respectively, as the sole nitrogen and carbon sources. Expression was induced with 1 mM IPTG and cells grown at 22 °C overnight.Cells were resuspended in 20 mM Tris HCl pH 8.0, 500 mM NaCl, 5 mM imidazole, 1 mM ß-mercaptoethanol, Complete® protease inhibitors EDTA-free (1 tablet/50 ml), 2 mM MgCl2, 10 units Benzonase/ml (> 90% purity Novagen) and broken by French Press. The soluble fraction was applied to an 8 ml MC-Poros column. Fractions containing eluted fusion protein were pooled, dialysed against 20 mM Tris pH 8.0, 300 mM NaCl, 1 mM ß-mercaptoethanol and simultaneously cleaved with TEV-protease at 15 °C.Removal of uncleaved protein and free His-tag was carried out by running the protein once more through the MC-Poros column under cleaving conditions. The flow-through containing the RGS18 domain with two additional residues (SM) at the N-terminus was concentrated to 1 mM and exchanged into an NMR buffer comprising 20 mM NaPi pH 6.0, 50 mM NaCl and 1 mM dDTT. Typically, 15 mg of RGS18 were purified from 1 l of culture. Protein molecular masses were confirmed by mass spectrometry.
NMR spectroscopy
NMR spectra were acquired at 297 K, using a Bruker DRX600 spectrometer in standard configuration with triple resonance probes equipped with self-shielded triple axis gradient coils and Bruker DRX600 and DMX750 spectrometers in standard configuration with triple resonance cryogenic probes equipped with self-shielded single axis gradient coils. Spectra for the resonance and NOE assignment were recorded essentially as described in the original references. A 1 mM 15N-labelled RGS18 sample in 90% H2O/10% D2O (NMR buffer; pH 6.0) was used for 3D 15N-separated NOESY-HSQC, 15N T1 and 15N T2 relaxation, and heteronuclear 15N-1H NOE experiments. A 1 mM 13C,15N-labelled sample of RGS18 in 90% H2O/10% D2O (NMR buffer; pH 6.0) was used for all HN-detected triple resonance experiments, 3D CBCA(CO)NNH, CBCANNH, CC(CO)NNH, H(CCCO)NNH, HBHA(CBCACO)NNH, HNCO, HN(CA)CO, and for a 3D 13C-separated, aliphatic-centred NOESY-HSQC spectrum. The sample was then freeze-dried and redissolved in 100% D2O for acquisition of 3D 13C-separated HMQC-NOESY, HCCH-COSY, HCCH-TOCSY and 2D NOESY and TOCSY spectra. Data were processed using the program XWIN-NMR (version 2.6) of Bruker BioSpin GmbH (Rheinstetten, Germany). Assignment of 13C, 15N and 1H resonances was carried out using standard assignment procedures on Silicon Graphics O2 workstations and an Intel Dual Xeon 3GHz PC, with the interactive program CCPNMR Analysis version 1.0.9 (Vranken et al., 2005).
Extent of Assignment and data deposition
Figure 1 shows the assigned 15N HSQC spectrum of RGS18 (Swissprot accession SP:Q9NS28). Backbone 1H, 13C and 15N resonances (including carbonyl carbons) were assigned for the first 133 residues of this 151 residue protein. The C-terminal 19 residues were not assigned, as their chemical shifts varied over time, and relaxation and heteronuclear NOE data showed these residues to be highly flexible. The high degree of overlap for the 11 Phe sidechains meant that 3 H, 4 H and 5 H resonances could not assigned unambiguously. Similarly, a high degree of overlap for the 5 Asn and 8 Gln sidechain NH2 sidechains resulted in only two unambiguous assignments being possible for these groups. Almost all remaining sidechain 13C and 1H resonances were assigned, including all Trp indole N1, H1 atoms and 2 of the 4 Arg sidechain N, H atoms which were not in the flexible C-terminal tail of the protein. The assignments are deposited in the BioMagResBank ( under accession code BMRB-7106.
Acknowledgements
The Structural Genomics Consortium is a registered charity (number 1097737) funded by the Wellcome Trust, GlaxoSmithKline, Genome Canada, the Canadian Institutes of Health Research, the Ontario Innovation Trust, the Ontario Research and Development Challenge Fund and the Canadian Foundation for Innovation. We thank Dr. Peter Schmieder and Dr. Christoph Brockmann for help with the NMR spectroscopy.
Figure Legend
Figure 1: Assigned 1H-15N-HSQC spectrum of human RGS18 recorded at 297 K and 750MHz.
References
Bernstein L. S., et al. (2000) Journal of Biological Chemistry275:18520-18526.
Chen C. H., et al. (1999) Journal of Biological Chemistry274:19799-19806.
Gagnon A. W., et al. (2002) Cellular Signalling14:595-606.
Nagata Y., et al. (2001) Blood97:3051-3060.
Yowe D., et al. (2001) Biochemical Journal359:109-118.
Shi G. X., et al. (2004) Journal of Immunology172:5175-5184.
Park I. K., et al. (2001) Journal of Biological Chemistry276:915-923.
Vranken W. F., et al. (2005) Proteins: Structure Function and Bioinformatics59:687-696.
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