Resonance Assignment of the RGS Domain of Human RGS10

Transcription

1 Resonance Assignment of the RGS Domain of Human RGS10 Oleg Y. Fedorov 2, Victoria A. Higman 1, Peter Schmieder 1, Martina Leidert 1, Annette Diehl 1, Jonathan Elkins 2, Meera Soundararajan 2, Hartmut Oschkinat 1 and Linda J. Ball 2 1 Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle Str. 10, Berlin. Germany. 2 Structural Genomics Consortium, University of Oxford, Botnar Research Centre, Oxford OX3 7LD. UK. RGS10 is a member of the D/R12 subfamily of Regulators of G-protein Signalling (RGS) proteins and acts as a GTPase-activating protein (GAP) via modulation of Gαi and Gαz signalling but does not interact with the structurally distinct Gαs subunit (Hunt et al., 1996). Unlike other subfamilies of RGS proteins, RGS10 lacks the N-terminal ampipathic helix that localizes these proteins to the membrane. Instead, membrane localization is regulated by palmitoylation of a conserved cysteine within the GTPase-activating RGS domain (Tu et al., 1999). 1 H, 15 N and 13 C resonance assignments of a construct containing the RGS domain from human RGS10 were obtained from 2D and 3D heteronuclear NMR spectra. The backbone assignment was completed with the exception of the first and last two residues and a highly overlapped stretch of five Glu residues ( of our construct). Sidechain assignment was 89% complete including 92 % of observable aromatics. The assignments were deposited with accession code BMRB References: Hunt T. W., et al. (1996) Nature 383: ; Tu Y. P., et al. (1999) J. Biol. Chem. 274: Character Count: 1487 (1500 max.)

2 K102 M59 Q60 N109 S7 Q54 W11 L126 S115 S122 7S I91 V81 E61 Y67 L124 Q78 Q84 R130 K112 K90 E132 Q58 K32 K62 N40 H96 K127 L89 D105 K64 M111 F28 V79 Q106 K74 K6 I107V25 F43 Q3 H128 C47 D114 E94 F36 F25 D49 E30 S14 R27 R118 K10 M53 G83 L19 T8 E35 L9 T69 T131 S85 S76 T57 Q101 L45 M68 E39 E16 F70 S72 K51 A12 L71 N17 L92 K52 V41 K34 S73 L89 D Q104 D21 L18 D123 N80 F L120 L32 A63 K121 E93 E20 R29 F100 Y Y113 S37 I103 M99 E65 F31 D55 W44 L110 A13 R86 F50 L42 I87 S117 G24 15N E23 S4 I66 A46 E38 L15 E82 E48 A9 A75 L5 F K56 W11e W44e H

3 Letter to the Editor: Backbone and Sidechain 1 H, 13 C and 15 N Resonance Assignment of the RGS Domain of Human RGS10 Oleg Y. Fedorov 2, Victoria A. Higman 1, Peter Schmieder 1, Martina Leidert 1, Annette Diehl 1, Jonathan Elkins 2, Meera Soundararajan 2, Hartmut Oschkinat 1 and Linda J. Ball 2. 1 Leibniz-Institut für Molekulare Pharmakologie (F.M.P.), Robert-Roessle Str., 10, D Berlin. Germany. 2 Structural Genomics Consortium (S.G.C), University of Oxford, Botnar Research Centre, Oxford OX3 7LD. UK. Corresponding author: Dr. L. J. Ball Structural Genomics Consortium (S.G.C), University of Oxford, Botnar Research Centre, Oxford OX3 7LD. UK. Tel: ; Fax: linda.ball@sgc.ox.ac.uk Keywords: chemical shift, G-protein signalling, NMR, RGS domain Abbreviations: RGS10: regulator of G-protein signalling; NaPi: sodium phosphate buffer; IPTG: isopropyl-beta-d-thiogalactopyranoside

4 Biological context Regulators of G-protein signalling (RGS) proteins share a conserved ~120 amino acid sequence termed the RGS domain, which is involved in the termination of signals 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 Gα and Gβγ subunits. In the inactive state, these exist in complex with the Gα GTPase in its inactive GDP-bound form. Following activation, GDP is replaced with GTP, which induces dissociation of the Gα-Gβγ complex, releasing active Gα and Gβγ proteins. Although Gα has an intrinsic GTPase activity, this is greatly enhanced by association with an RGS domain. In this way, RGS proteins act as GTPase-activating proteins (GAPs). Following GTP hydrolysis, the resultant GDP-Gα complex can recombine with Gβγ to terminate the signal RGS10 is a member of the D/R12 subfamily of RGS proteins. It acts as a GTPase-activating protein (GAP) via modulation of Gαi and Gαz signalling but does not interact with the structurally and functionally distinct Gαs subunit (Hunt et al., 1996). Activity on Gαz is inhibited by palmitoylation of the G-protein (Tu et al., 1997). Unlike the R4 subfamilies of RGS proteins, RGS10 lacks the N-terminal ampipathic helix that localizes these proteins to the membrane (Bernstein et al., 2000;Chen et al., 1999). Instead, membrane localization of RGS10 is regulated by palmitoylation of a conserved cysteine residue within its RGS domain (Castro-Fernandez et al., 2002;Tu et al., 1999). Methods and experiments Cloning, expression and purification of human RGS10 The RGS domain of human RGS10 (residues Q16-L151 of RGS10_human, (Genbank gi: ; Swissprot O43665) plus two additional N-terminal residues (SM) from the vector was cloned into a homemade vector plic-sgc1, which adds a TEV-cleavable hexahistidine tag to the N-terminus. Uniformly 15 N- and 15 N, 13 C-labelled His-TEV-SM-RGS10 RGS 2

5 domains were prepared from E. coli BL21(DE3)-Rosetta cells (Novagen) containing this plasmid, grown in M9 minimal medium supplemented with 60 µg/ml carbenicillin, 30µg/ml chloramphenicol, and containing 0.5 g/l 15 NH 4 Cl and either 2 g/l (w/v) 12 C 6 -glucose or 13 C 6 - glucose, as the sole nitrogen and carbon sources, respectively. 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, 300 mm NaCl, 5 mm imidazole, 1 mm ß-mercaptoethanol, Complete EDTA-free protease inhibitor cocktail (Roche) and 10 units Benzonase/ml (> 90% pure; Novagene) 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 re-running the protein over the MC-Poros column under cleaving conditions. The flow-through containing the RGS domain was concentrated in NMR buffer comprising 20 mm NaPi ph 6.0, 50 mm NaCl and 1 mm ddtt. Typically, 14 mg of RGS domain were purified from 1 L of culture. Protein molecular weights were confirmed by mass spectrometry. NMR spectroscopy NMR spectra were acquired at 297 K, on Bruker DRX 600 and DMX 750 spectrometers in standard configuration with triple resonance probes equipped with self-shielded triple axis gradient coils. Spectra were recorded essentially as described in the original references. A 1.9 mm 15 N-labelled protein sample in 90% H 2 O/10% D 2 O (NMR buffer; ph 6.0), was used for 3D 15 N-separated NOESY-HSQC, 15 N T 1 and 15 N T 2 relaxation, and heteronuclear 15 N- 1 H NOE experiments. A 1 mm 13 C, 15 N-labelled sample in 90% H 2 O/10% D 2 O (NMR buffer; ph 6.0) was used for all H N -detected triple resonance experiments, 3D CBCA(CO)NNH, CBCANNH, CC(CO)NNH, H(CCCO)NNH, HBHA(CBCACO)NNH, HNCO, HN(CA)CO, and for 3D 13 C-separated, aliphatic-centred and aromatic-centred NOESY-HSQC spectra. This sample was freeze-dried and redissolved in 100% D 2 O for acquisition of 3D 13 C- separated HMQC-NOESY, HCCH-COSY, HCCH-TOCSY and 2D NOESY, 2D TOCSY 3

6 spectra. Data were processed using the program XWIN-NMR (version 2.6) of Bruker BioSpin GmbH (Rheinstetten, Germany). Assignment of 13 C, 15 N and 1 H resonances was carried out using standard assignment procedures on a Linux workstation (Intel Dual Xeon 3GHz PC), with the interactive program CCPNMR Analysis version (Vranken et al., 2005). Extent of Assignment and data deposition Figure 1 shows the assigned 15 N HSQC spectrum of the RGS domain from human RGS10. All backbone 1 H, 13 C and 15 N resonances (including carbonyl carbons) were assigned with the exception of the first and last two residues and a sequence segment of five Glu residues (residues ; numbering relative to our construct) where peaks were obscured due to spectral overlap. A high degree of overlap for the Phe sidechains also meant that two Hδ, three Hε and five Hζ resonances could not be assigned unambiguously. The Asn and Gln sidechain NH 2 sidechains were all assigned with the exception of Gln104. Almost all remaining sidechain 13 C and 1 H resonances were assigned, including Trp11 and Trp44 indole Nε1 and Hε1 atoms. No Arg sidechain Nε, Hε atom assignments were possible. The assignments were deposited in the BioMagResBank ( under accession code BMRB Acknowledgements The Structural Genomics Consortium is a registered charity (number ) 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 1 H- 15 N-HSQC spectrum of the RGS domain from human RGS10 recorded at 297 K and 750 MHz. 4

7 References Hunt T. W., et al. (1996) Nature 383: Tu Y. P., et al. (1997) Science 278: Bernstein L. S., et al. (2000) Journal of Biological Chemistry 275: Chen C. H., et al. (1999) Journal of Biological Chemistry 274: Castro-Fernandez C., et al. (2002) Endocrinology 143: Tu Y. P., et al. (1999) Journal of Biological Chemistry 274: Vranken W. F., et al. (2005) Proteins: Structure Function and Bioinformatics 59: