June 29, 2010
I suppose you’ve all seen cartoons of GPCR structures, snake plots and the like. Isn’t it odd that the orientations of most GPCRs are always shown as N-Terminus “up”, and with all other TM7 membrane proteins, such as bacteriorhodopsin and bovine rhodopsin – a bona fide GPCR – it’s vice versa? In other words, all GPCR models, with the distinct exception of rhodopsin, imply that the space below the horizontal virtual membrane is inside the cell and above the membrane is outside. Like so:
You're doing it wrong! Left: beta2Adrenergic Receptor with extracellular domains pointing up. Right: Bovine Rhodopsin: Blue carbohydrate extracellular domain pointing down indicating the location of 'outside the cell' = down.
How did that happen?
I don’t really know. But I do know that it’s been a nuisance and this lack of convention is becoming even worse now that the internal workings of GPCRs can be seen at sub-Angstrom resolution. It makes it difficult to quickly ‘get it’, because of these opposing canonical views.
Here’s what I think where this messy situation originated from: pharmacologists and biologists think of the receptor extracellular portion pointing ‘up’ and towards them as they lean over their cell culture dish that is filled with cells expressing the receptor under investigation. Most of these experiments involve the addition of ligands at some point and these are often added with a pipettor that reaches a layer of cells from the top. Topologically, these researchers are situated ‘outside’ of the cells they work with. So, to pharmacologists and biologists it makes sense that GPCR extracellular domains point ‘up’, because they interfere with from the ‘outside’, the N-terminus, extracellular moiety of the receptor.
Physicists however, have a tradition to working with samples that are simple to deal with, such as Purple Membrane with bacteriorhodopsin or bovine rhodopsin. This is because these 7TM proteins are activated by light and physicists know really well how to work with photons. Now, when you apply these membranes to a measurement device, for example onto the substrate of an infra red instrument, you do this with your pipettor by hand and the the action is taking place where the topologically open membranes meet that solid surface – presumably with the 7TM ‘busy end’, the extracellular moiety, pointing towards the instrument:
Does that make any sense? The 'business end' of GPCRs receptors are perceived as pointing towards biologists/pharmacologists as they apply reagents. When Phycisists add membrane preps of somewhat simpler do deal with 7TM proteins they pay attention of the "business end" pointing towards the detection hardware.
So, it could be a matter of perspective employed by physicists and biologists/pharmacologists that lead to this unfortunate lack of convention.
Maybe not. I’d love to hear about more plausible explanations!
Cheers,
Peter
P.S. What’s your preferred way to look at a GPCR? Sorry, there are no polls available at the moment.
Filed under:
Membrane Proteins, Opinion, Structure | Posted by: Peter Nollert | Comments: 7
June 29, 2010
So far eight different types of GPCR structures have been determined by X-ray crystallography: β2 Adrenergic Receptor, β1 Adrenergic Receptor, Adenosine Receptor, Rhodopsin, CXCR4 Chemokine receptor, Dopamine D3 receptor, the Histamine Receptor and the S1P1 Sphingosine 1-phosphate receptor.
Updates:
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7 Oct 2010: added CXCR4 Chemokine Receptor
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18 Nov 2010: added Dopamine D3 Receptor
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The tables below makes it easier to find them either as a PDB code or the original reference.
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21 Jan 2011: β1 and β2 Adrenergic Receptor structures
stabilized active state of β2 Adrenergic Receptor: 3P0G with irreversible agonist: 3PDS partial agonist and agonist structures of β1 2y00, 2Y01, 2Y02, 2Y03, 2Y04
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27 March 2011: A2A Adenonsine Receptor with bound agonist: 3QAK
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30 March 2011: Dobutamin bound beta 1 adrenergic receptor (turkey): 2Y01
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22 June 2011: Histamine H1 Recepotor bound to doxepin: 3RZE ; A2A Adenosine Receptor with bound agonists Adenosine 2YDO & NECA 2YDV
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20 July 2011: agonist-occupied β2 adrenergic receptor (active) in complex with the (nucleotide free) Gs heterotrimer: 3SN6
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17 Feb 2012: sphingosine 1-phosphate receptor 1 (S1P1-T4L) with a bound sphingolipid mimic (antagonist): 3v2W
If there's a new GPCR structure out there and it's not on this list: drop me a line so I can included it here.
Thanks!
Peter
Histamine H1 Receptor
| Structure Notes | PDB | Resolution [A] | Reference |
| H1R with bound drug molecule doxepin | 3RZE | 3.1 | T.Shimamura, M. Shiroishi, S. Weyand, H.Tsujimoto, G. Winter, V. Katritch, R. Abagyan, V. Cherezov, W. Liu, G.W. Han, T. Kobayashi, R.C. Stevens & So Iwata
Structure of the human histamine H1 receptor complex with doxepin
Nature (2011) doi:10.1038/nature10236 |
Sphingosine 1-phosphate Receptor
| Structure Notes |
PDB |
Resolution [A] |
Reference |
| sphingosine 1-phosphate receptor 1 (S1P1-T4L) with a bound sphingolipid mimic (antagonist) |
3V2W |
3.35 |
Crystal Structure of a Lipid G Protein–Coupled Receptor
Michael A. Hanson, Christopher B. Roth, Euijung Jo,Mark T. Griffith, Fiona L. Scott, Greg Reinhart, Hans Desale, Bryan Clemons, Stuart M. Cahalan, Stephan C. Schuerer, M. Germana Sanna, Gye Won Han, Peter Kuhn, Hugh Rosen, Raymond C. Stevens
Science Vol. 335 no. 6070 pp. 851-855; 2012 |
Dopamine D3 Receptor
| Structure Notes |
PDB |
Resolution [A] |
Reference |
| D(3) dopamine receptor, T4 lysozyme insertion in 3rd intracellular loop, in complex with Eticlopride, crystallized in LCP (Cholesterol additive) |
3PBL |
2.9 A |
No publication yet. PDB authors: Chien, E.Y.T., Liu, W., Han, G.W., Katritch, V., Zhao, Q., Cherezov, V., Stevens, R.C., Accelerated Technologies Center for Gene to 3D Structure (ATCG3D) |
Rhodopsin
| Structure Notes |
PDB |
Resolution [A] |
Reference |
| first experimental GPCR structures |
1F88, 1HZX |
2.8 |
Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M. Science. 2000 Aug Crystal structure of rhodopsin: A G protein-coupled receptor. 4;289(5480):739-45.
Teller DC, Okada T, Behnke CA, Palczewski K, Stenkamp RE.
Advances in determination of a high-resolution three-dimensional structure of rhodopsin, a model of G-protein-coupled receptors (GPCRs). Biochemistry. 2001 Jul 3;40(26):7761-72. |
| shows functional water molecules |
1L9H |
2.6 |
Okada T, Fujiyoshi Y, Silow M, Navarro J, Landau EM, Shichida Y.
Functional role of internal water molecules in rhodopsin revealed by X-ray crystallography.
Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):5982-7. Epub 2002 Apr 23. |
| focus on retinal conformation |
1U19 |
2.2 |
Okada T, Sugihara M, Bondar AN, Elstner M, Entel P, Buss V. The retinal conformation and its environment in rhodopsin in light of a new 2.2 A crystal structure. J Mol Biol. 2004 Sep 10;342(2):571-83. |
| Thermostable N2C/D282C mutant heterologously expressed in COS cells |
2J4Y |
3.4 |
Standfuss J, Xie G, Edwards PC, Burghammer M, Oprian DD, Schertler GF. Crystal structure of a thermally stable rhodopsin mutant. J Mol Biol. 2007 Oct 5;372(5):1179-88. Epub 2007 Mar 12. |
| photoactivated and ground state |
2I35, 2I36, 2I37 |
3.8, 4.1, 4.15 |
Salom D, Lodowski DT, Stenkamp RE, Le Trong I, Golczak M, Jastrzebska B, Harris T, Ballesteros JA, Palczewski K. Crystal structure of a photoactivated deprotonated intermediate of rhodopsin. Proc Natl Acad Sci U S A. 2006 Oct 31;103(44):16123-8. Epub 2006 Oct 23 |
| Retinal removed: Opsin |
3CAP |
2.9 |
Park JH, Scheerer P, Hofmann KP, Choe HW, Ernst OP. Nature. 2008 Jul 10;454(7201):183-7. Epub 2008 Jun 18. Crystal structure of the ligand-free G-protein-coupled receptor opsin. |
| activated form of Ops*-GalphaCT peptide complex |
3DQB |
3.2 |
Scheerer P, Park JH, Hildebrand PW, Kim YJ, Krauss N, Choe HW, Hofmann KP, Ernst OP. Crystal structure of opsin in its G-protein-interacting conformation. Nature. 2008 Sep 25;455(7212):497-502. |
| Squid Rhodopsin |
2Z73 |
2.5 |
Murakami M, Kouyama T. Crystal structure of squid rhodopsin. Nature. 2008 May 15;453(7193):363-7. |
CXCR4 Chemokine Receptor
| Structure Notes |
PDB |
Resolution [A] |
Reference |
| complex with small molecule antagonist IT1t and cyclic peptide antagonist CVX15, T4 lysozyme insertion in 3rd intracellular loop, stabilizing mutations, crystallized in LCP (Cholesterol additive) |
3ODU, 3OE0, 3OE8, 3OE9, 3OE6 |
2.5 A, 2.9 A, 3.1 A, 3.1 A, 3.2 A |
Wu, B. et al., Structures of the CXCR4 Chemokine GPCR with Small-Molecule and Cyclic Peptide Antagonists. Science 7 Oct 2010 |
β1 Adrenergic Receptor
| Structure Notes |
PDB |
Resolution [A] |
Reference |
| Dobutamin bound beta 1 adrenergic receptor (turkey) |
2Y01 |
2.6 |
Warne, A. et al. (2011) TURKEY BETA1 ADRENERGIC RECEPTOR WITH STABILISING MUTATIONS AND BOUND PARTIAL AGONIST DOBUTAMINE (CRYSTAL DOB102) Nature 469: 241 |
| Thermostabilized turkey receptor |
2VT4 |
2.7 |
Warne T. et al., (2008) Structure of a beta1-adrenergic G-protein-coupled receptor
Nature 454, 486-491 |
Adenosine Receptor
| Structure Notes |
PDB |
Resolution [A] |
Reference |
| Bound antagonist ZM241385 |
3EML |
2.6 |
Jaakola VP, Griffith MT, Hanson MA, Cherezov V, Chien EY, Lane JR, Ijzerman AP, Stevens RC. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science. 2008 Nov 21;322(5905):1211-7. Epub 2008 Oct 2. |
| Bound agonist UK-432097 |
3QAK |
2.7 |
Structure of an Agonist-Bound Human A2A Adenosine Receptor
Xu, F., Wu, H., Katritch, V., Han, G.W., Jacobson, K.A., Gao, Z-D., Cherezov, V., Stevens, R.C.
Science DOI: 10.1126/science.1202793 |
| Bound to agonists adenosine and NECA |
2YDO 2YDV |
3.0 & 2.6 |
G. Lebon, T. Warne, P. C. Edwards, K. Bennett, C. J. Langmead, A. G. W. Leslie & C. G. Tate
Agonist-bound adenosine A(2A) receptor structures reveal common features of GPCR activation
Nature 474, 521–525 (23 June 2011) |
| A2A adrenergic receptor bound to Fab2839 |
3VG9
3VGA |
3.1 & 2.7 |
Hino, T., Arakawa, T., Iwanari, H., Yurugi-Kobayashi, T., Ikeda-Suno, C., Nakada-Nakura, Y., Kusano-Arai, O., Weyand, S., Shimamura, T., Nomura, N., Cameron, A., Kobayashi, T., Hamakubo, T., Iwata, S., & Murata, T. (2012). G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody |
β2 Adrenergic Receptor
| Structure Notes |
PDB |
Resolution [A] |
Reference |
| b2AR365-Fab5 complex |
2R4S, 2R4R |
3.4 / 3.4 |
Rasmussen SG, Choi HJ, Rosenbaum DM, Kobilka TS, Thian FS, Edwards PC, Burghammer M, Ratnala VR, Sanishvili R, Fischetti RF, Schertler GF, Weis WI, & Kobilka BK (2007). Crystal structure of the human β2 adrenergic G-protein-coupled receptor. Nature 450:383-387. |
| Complex with Carazolol ligand and bound Cholesterol; T4 lysozyme fusion in 3rd intracellular loop |
2RH1 |
2.4 |
Cherezov et al. (2007). High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 318:1258-1265 |
| T4 lysozyme fusion in 3rd intracellular loop, bound cholesterol |
3D4S |
2.8 |
Hanson et al., (2008)
A specific cholesterol binding site is established by the 2.8 A structure of the human beta2-adrenergic receptor. Structure 16: 897-905 |
| methylated receptor |
3KJ6 |
3.4 |
Bokoch et al., (2010) Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor.
(2010) Nature 463: 108-112 |
| T4 lysozyme fusion in 3rd intracellular loop, bound cholesterol, mutations: E122W, N187E, C1054T, C1097A; inverse agonist ICI 118,551 |
3NY8 |
2.84 |
To be Published: Crystal structure of the human beta2 adrenergic receptor in complex with the inverse agonist ICI 118,551 |
| T4 lysozyme fusion in 3rd intracellular loop, bound cholesterol, mutations: E122W, N187E, C1054T, C1097A; Timolol |
3NY9 |
2.48 |
To be published: Crystal structure of the human beta2 adrenergic receptor in complex with a novel inverse agonist |
| T4 lysozyme fusion in 3rd intracellular loop, bound cholesterol, mutations: E122W, N187E, C1054T, C1097A; antagonist alprenolol |
3NYA |
3.16 |
Conserved binding mode of human beta2 adrenergic receptor inverse agonists and antagonist revealed by X-ray crystallography |
| agonist-occupied β2 adrenergic receptor (active) in complex with the (nucleotide free) Gs heterotrimer |
3SN6 |
3.2 |
Rasmussen et al.
Crystal structure of the β2 adrenergic receptor–Gs protein complex
Nature (2011) |
Filed under:
Membrane Proteins, Crystallization, Lists, Structure | Posted by: Peter Nollert | Comments: 10
June 28, 2010
With the first medically relevant GPCR structures published about 2 years ago, we can now see, for a few select examples, the 'inner workings' of GPCRs in great detail. The key to all of this was of course sample preparation, and that is: production of GPCR samples in sufficient quantity and quality and then their crystallization into well-ordered protein crystals. The fact that we can visualize GPCR molecules on an atom scale is due to identifying appropriate crystallization conditions for the respective target. Below is a compilation with the described crystallization conditions.
More to come ;)
Peter
|
GPCR Target and Crystallization Technique |
Protein Sample |
Precipitation Reagent |
TimeTemp. |
PDB, Resolution, Ref. |
|
human β2 adrenergicreceptor, b2AR365-Fab5 complex crystallized in DMPC/CHAPSO bicelles; hanging drop vapour diffusion |
8-12 mg/ml of b2AR-Fab5 complex; concentrated to 60 mg/ml in 10 mM HEPES pH 7.5, 100 mM NaCl, 0.1 % Dodecylmaltoside, 10 uM carazolol |
Ammonium Sulphate, sodium acetate and EDTA, pH 6.5-7.5 |
7-10 days |
2R4S 3.4A REF |
|
human β2 adrenergicreceptor complex with Carazolol ligand and bound Cholesterol crystallized within Lipidic Cubic Phase; receptor with T4 lysozyme fusion in 3rd intracellular loop |
In Monoolein-based lipidic cubic phase, doped with ca. 8-10% Cholesterol; portions of 25-50 nL of protein-laden LCP were overlaid with 0.8uL of precipitant solution |
30-35%(v/v) PEG400, 100-200 mM NaSulfate, 100mM Bis-tris propane pH 6.5-7.0, 5-7%(v/v) 1,4-butanediol |
21-23 C |
2RH1 2.4A REF |
|
human β2 adrenergicreceptor receptor with T4 lysozyme fusion in 3rd intracellular loop; Lipidic cubic phase based crystallization; receptor bound to Cholesterol and Timolol |
Crystals grown in25 nL or 50 nL portions of a 10% w/w cholesterol spiked monoolein-based lipidic cubic phase. Receptor concentrated to 30 mg/mL. |
0.8 uL overlaying solution: 28% w/v PEG 400, 300 mM K Formate, 100 mM Bis-tris propane pH 7.0 and 2 mM timolol |
|
3D4S 2.8A REF |
|
Turkey β1 Adrenergic Receptor containing stabilizing mutations and bound Cyanopindolol crystallized by traditional vapor diffusion from micellar solution |
Protein concentration 6 mg/ml |
10 mM Tris-HCL, pH 7.7; 50 mM NaCl, 0.1 mM EDTA, 0.35% Octylthiglucoside, 0.5 mM Cyanopindolol against reservoir filled with 100 mM ADA pH 6.9-7.3; 29-32% PEG600 |
|
2VT4 2.7A REF |
|
Human A2a Adenonsine Receptor with T4lysozyme in 3rd intracellular loop, Lipidic cubic phase based crystallization |
Lipidic matrix consisting of Monoolein and Cholesterol; receptor concentrated to 70 mg/ml; In Monoolein-based lipidic cubic phase, doped with ca. 12% Cholesterol; portions of 50 nL of protein-laden LCP were overlaid with 0.8uL of precipitant solution |
30% (v/v) PEG 400 (range of 28-32%), 186 mM Lithium sulfate (range of 180 to 220 mM), 100 mM Sodium citrate (pH 6.5) (Range of 5.5 to 6.5) and 200 μM ZM241385 |
21-23 C |
3EML 2.6A Science, Science supp. |
|
Methylated beta 2 Adrenergic Receptor-Fab complex |
methylated receptor |
|
|
3KJ6 3.4A REF |
Filed under:
Membrane Proteins, Crystallization | Posted by: Peter Nollert | Comments: 0
June 24, 2010
Where do you search for exotic variants of a particular GPCR? How do you identify natural sequence variants of a particular receptor? And how about matching a receptor with a particular synthetic ligand, or the other way around? This is where I'd start:
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IUPHAR Database Comprehensive listing of GPCRs with information related to genes and ligands that bind particular receptors; listed chronologically with receptor/ligand pairings.
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GLIDA: GPCR-Ligand Database Information on GPCRs and their known ligands. Start search with either GPCR or ligand. Last entry: 10/10/2009.
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GPCRDB A comprehensive, sequence focused information system for G protein-coupled receptors. Browse GPCR families interactively, create web-based blast, alignments and predict the effect of point mutations.
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gpDB A database of GPCRs, G-proteins, Effectors and their interactions. Last update: 20/03/2008.
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Endogenous GPCRs in common cell lines Useful for double-checking heterologous receptor expression.
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GPCR Natural Variants Database Describes sequence variants of GPCRs in humans.
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GPCR Pattern Recognition Tool to query a sequence against ca. 120 GPCR fingerprints
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SEVENS GPCR gene information on a genomic scale.
Are there any other databases or online resources that should be included here?
Thanks! And a special thanks to the curators of these useful GPCR database tools.
Peter
Filed under:
Membrane Proteins, Online Tools, Sequence | Posted by: Peter Nollert | Comments: 1