October 10, 2012
Nobel prize for GPCRs!
Congratulations to Brian Kobilka and Jeff Lefkowitz for the 2012 Chemistry Nobel Prize !
Now the world will need to learn how to say 'G-protein coupled receptors'.
C O N G R A T U L A T I O N S ! ! ! !
Emerald Insights Blog: Membrane Proteins
April 2, 2012
Membrane Protein Structure Databases
As interest in membrane protein targets by both academic laboratories and large pharmacological companies grows, becoming versed in the topic of membrane proteins is more important than ever. After working with soluble proteins, the world of membrane protein structure can be intimidating to the novice structural biologist just entering the field. With over 80,000 structures present in the Protein Data Bank, searching through this vast number of entries for membrane protein structures can be akin to searching for a needle in a haystack. In this post I will highlight and discuss the features of three repositories of membrane protein structures: Membrane Proteins of Known 3D Structure, Membrane Protein Databank, and Protein Databank of Transmembrane Proteins.
The Membrane Proteins of Known 3D Structure database is curated by the lab of Dr. Stephen White at the University of California Irvine. The database is updated often, well-maintained, and contains structures of membrane proteins determined by both X-ray and electron diffraction methods (although you’ll find a few NMR structures in there as well). Membrane protein structures are divided into three classes on the site: (1) monotopic transmembrane proteins, (2) multi-pass beta-barrel transmembrane proteins, and (3) multi-pass alpha-helical transmembrane proteins. Each class of protein is the further sub-classified into functional groups via a drop-down menu. So if I wanted to look at available structures for autotransporters (my personal favorite), I would open the “Transmembrane Proteins: Beta-Barrel” drop-down menu and then select “Outer Membrane Autotransporters”. Proteins that have multiple entries (ie: different ligands bound) are then grouped together so you don’t have to go searching through a long list to find them all. The database is also accessible using text-based searches. At the time of writing of this post there were 956 entries in this database, 322 of those being unique.
The Membrane Protein Databank was started by the lab of Dr. Martin Caffrey, currently at the University of Limerick in Ireland. It is updated weekly and consists of membrane protein structures determined by X-ray diffraction, electron diffraction, NMR, and cryoelectron miscroscopy. The database is searchable by a number of different criteria including but not limited to: expression system, function, journal of publication, ligand, pH, resolution, alpha-helical vs. beta-sheet, crystallization method, and temperature. In addition, the statistics function makes doing searches like “How many structures have been solved using bicelles?” a breeze to answer. Simply select Crystallization Method for “Statistics on Membrane Protein Versus”, All Experimental Techniques for “As Appropriate, Limit Analysis by Experimental Technique”, and Bicelle for “As Approriate, Limit Analysis by Crystallization Method” and voila! At the time of the writing of this post the MPDB database held 1096 entries.
The Protein Data Bank of Transmembrane Proteins is maintained by the Institute of Enzymology in Hungary. The database is updated by an automated algorithm called TMDET that scans the entire PDB every week. This database is searchable by PDB code, PDB keyword, alpha-helical vs. beta sheet, and number of transmembrane segments. For example, if I wanted to ask the question “How many beta-barrel proteins structures exist that have 22 strands present?”, I would simply pick beta-barrels as the search type and then 22 as the number of transmembrane segments. This will return a list of 22-stranded beta-barrels with links to download the PDB text file for each entry. At the time of the writing of this post the TMDET algorithm has identified what it believes to be 1568 membrane proteins, 1348 of those alpha-helical and 219 of them beta-barrels.
References 
- Raman, P., Cherezov, V., & Caffrey, M. (2005). The Membrane Protein Data Bank Cellular and Molecular Life Sciences, 63 (1), 36-51 DOI: 10.1007/s00018-005-5350-6
- Tusnády GE, Dosztányi Z, & Simon I (2004). Transmembrane proteins in the Protein Data Bank: identification and classification. Bioinformatics (Oxford, England), 20 (17), 2964-72 PMID: 15180935
March 22, 2012
Two morphine receptor structures: explaining human behavior from atoms up
We've almost gotten used to GPCR structure publications - but today I'm seeing two publications in Nature that ought to rock the world of GPCR structural biology: morphine receptors. Structures of antagonist bound µ-opioid receptor and κ-opioid receptor with bound antagonist. The impact of these structures can not be underestimated since these structures give insight into the most used (and abused) clinical drugs and their workings on an atom scale. In a nutshell, these structures connect atom scale molecular structure to human behavior.
Manglik, A., Kruse, A., Kobilka, T., Thian, F., Mathiesen, J., Sunahara, R., Pardo, L., Weis, W., Kobilka, B., & Granier, S. (2012). Crystal structure of the µ-opioid receptor bound to a morphinan antagonist Nature DOI: 10.1038/nature10954
Wu, H., Wacker, D., Mileni, M., Katritch, V., Han, G., Vardy, E., Liu, W., Thompson, A., Huang, X., Carroll, F., Mascarella, S., Westkaemper, R., Mosier, P., Roth, B., Cherezov, V., & Stevens, R. (2012). Structure of the human κ-opioid receptor in complex with JDTic Nature DOI: 10.1038/nature10939
Congratulations to the Kobilka and the Stevens teams!
February 18, 2012
New and Different GPCR Binding Pockets
What a staggering variety of access to GPCR binding pockets there is. The recently published GPCR structures shed new light on the exquisite architecture of GPCR binding pockets:
1. An internal hydrophobic binding pocket that is pretty much closed towards the aqueous phase was identified in the crystal structure of the sphingosine 1-phosphate receptor 1 (S1P1-T4L) with a bound sphingolipid mimic (antagonist). Similar to rhodopsin, the ligand can access to the deep binding cavity from the hydrophobic section of the membrane.
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
2. In addition to this conventional ligand binding location, there seems to be a site close to the intracellular surface of GPCRs that serves as an alternate way to modulate GPCR activity in A2A adrenergic receptor and unrelated GPCRs. The structure of the A2A adenosine receptor with a Fab fragment (Fab2839) reveals how it penetrates the receptor with its CDR-H3 domain. The crux is that the interaction is similar to that of the activated b2-adrenergic receptor and that of opsin with a bound peptide; interestingly, the binding of the Fab fragment inactivates the A2A adenosine receptor. This discovery could have huge ramifications, since it offers an alternate way to modulate GPCR activity, without occupying the standard central ligand binding pocket.
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 Nature DOI:10.1038/nature10750
All this news is phantastic! The more we look the more we see.
Cheers,
Peter
January 31, 2012
M2 Muscarinic Acetylcholine Receptor Structure
Congratulations to the publication of the first GPCR structure in 2012! These congrats go to the Kobilka, Haga and Kobayashi teams that reported the X-ray crystallographic structure of the M2 muscarinic acetylcholine receptor this weeks' issue of Nature. The bound antagonist (3-quinuclidinyl-benzilate) is bound in the center of a long aqueous tubular structure that has not seen before in any GPCR. Apart from the common arrangement of the 7 TMs, the comparison of the shape of the M2 binding pockets with those of other receptors (b2, A2A, CXCR4, D3 and H1 receptor) shows that the binding modes are very different from each other (as compared to b2, A2A, CXCR4, D3 and H1 receptors, see Fig.4 here). The ligand binding pocket is really a channel that goes two thirds through the membrane, and opening up the remainder would likely convert this GPCR into a water pore. Turns out that the M2 binding pocket is very similar amongst the family of muscarinic acetylcholine receptors, explaining the difficulty of developing specific ligands.
As seen with many other GPCR structures, T4 lysozyme inserting into the third intracellular loop provides for strong packing interactions between layers in the M2-T4L crystal. No surprise here. As of writing this, the coordinates are not yet available (access code 3UON) but should be released soon.
Cheers,
Peter
PS: More GPCR structures to come: e.g. the yet unpublished S1P1 and k-opioid receptor structure are advertised here
November 7, 2011
Comparing Apples with Oranges: B&W
Identifying individual amino acid residues within a GPCR and comparing these across different receptors is a routine task that’s helped by a widely accepted nomenclature system: that of Ballesteros and Weinstein.
Juan A. Ballesteros, Harel Weinstein (1995). Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors Methods in Neurosciences, 25, 366-428 DOI: 10.1016/S1043-9471(05)80049-7
You could look up the nomenclature rules in the original paper or find the rules online and apply them to the particular amino acid sequence you’re working with. This is a bit cumbersome, isn’t it?
Good news: here’s a simple way to check the Ballesteros&Weinstein nomenclature with the Sequence Tool provided by http://www.gpcr.org. Just call up the target sequence and hover over a particular amino acid to extract the Ballesteros&Weinstein(B&W) code:
A simple way to call up the Ballesteros&Weinstein(B&W) code for a particular amino acid in a GPCR target
I like simple.
Peter
November 7, 2011
More Options Than I Thought: GPCR Expression Systems
When it comes to expression systems, insect cells have been the primary supplier of GPCR protein for crystallographic studies. Notable exceptions are:
- Rhodopsins can be obtained from the retina of eyes from Squid or cows and alternatively be expressed in COS cells.
- The Histamine H1 receptor expressed in Pichia pastoris was used to determine its crystal structure
To attendees and listeners of the "GPCR expression for biophysical and structural studies" webinar: There was a question at the very end of the webinar and I'd like to correct the answer that I gave. While it is true that insect cells are an important source for heterologous expression of GPCRs, they're not the sole source of GPCR material for crystallization.
Histamine 1 receptor, expressed in Pichia pastoris as a fusion protein with Lysozyme as described in
Shimamura T, Shiroishi M, Weyand S, Tsujimoto H, Winter G, Katritch V, Abagyan R, Cherezov V, Liu W, Han GW, Kobayashi T, Stevens RC, & Iwata S (2011). Structure of the human histamine H1 receptor complex with doxepin. Nature, 475 (7354), 65-70 PMID: 21697825
Thanks to the listeners for making me aware of this!
Peter
October 11, 2011
Webinar on GPCR Expression for Biophysical and Structural Studies
GPCR Expression for Biophysical and Structural Studies
Date: Oct 18, 2011
Time: 11:00 – 11:50 AM PDT
Speaker: Peter Nollert, Ph.D.
GPCRs (G-protein coupled receptors) play a critical role in cellular signaling at the cell membrane. Although this class of transmembrane proteins has long been identified as a premier drug target, the availability of samples in quantities and qualities that are sufficient for biophysical and structural studies is often limited. Recent advances in structural and biophysical characterization for a select set of GPCRs have demonstrated that these proteins are indeed amenable to such studies. What are the tools that have enabled the preparation of GPCRs for such studies? This webinar provides insight into the latest advances in methods and techniques that Emerald Biostructures and other membrane protein experts have employed for the expression of this important target molecule class for structural and biophysical methods.
To join this free webinar, please sign up here.
https://www1.gotomeeting.com/register/473502641
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In case you have missed the webinar, here’s the recording:
GPCR expression webinar. Methods & techniques for expression of GPCRs for structural biology & biophysical methods
July 21, 2011
Relaying the Signal Inside the GPCR-Gs Complex: Breakthrough Structure (Ligand-β2AR-Gs)
If you want to witness scientific history in the make, it's been very rewarding to watch the discoveries in GPCR structural biology unfold over the past few months. This development is trumped today by the publication of the crystal structure of the agonist-occupied β2 adrenergic receptor (active) in complex with the (nucleotide free) Gs heterotrimer.
Rasmussen, S., DeVree, B., Zou, Y., Kruse, A., Chung, K., Kobilka, T., Thian, F., Chae, P., Pardon, E., Calinski, D., Mathiesen, J., Shah, S., Lyons, J., Caffrey, M., Gellman, S., Steyaert, J., Skiniotis, G., Weis, W., Sunahara, R., & Kobilka, B. (2011). Crystal structure of the β2 adrenergic receptor–Gs protein complex Nature DOI: 10.1038/nature10361
It would be an understatement to call this a great achievement.
This structure is C O L O S S A L . It shows in exquisite detail the conformational changes as they are propagated between the GPCR and the nucleotide-binding pocket of the G-protein. How does it work? In its agonist bound form the β2 adrenergic receptor can splay one helix, TM6, resulting in a whooping 14 Angstroms displacement of the helix termini with respect to that of a neighboring helix (TM4). And this in turn displaces a helix in Gαs; and now get this: this displacement takes place in a region that has the GTPase activity.
Another milestone structure that's been spearheaded in Brian Kobika's lab.
This is phenomenal. Congratulations!
Peter
June 23, 2011
Structures of Histamine H1 Receptor with Bound Drug Molecule and Adenosine Receptor with Bound Agonists
In a rather remarkable chain of two events, NATURE has published two GPCR structure papers: Histamine H1 Recepotor bound to doxepin: 3RZE and A2A Adenosine Receptor with bound agonists Adenosine 2YDO & NECA 2YDV.
Lebon G, Warne T, Edwards PC, Bennett K, Langmead CJ, Leslie AG, & Tate CG (2011). Agonist-bound adenosine A(2A) receptor structures reveal common features of GPCR activation. Nature PMID: 21593763
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
The GPCRs of known structures page has been updated accordingly.
My congratulations go to So Iwata and Chris Tate.
This is amazing,
Peter