New and different GPCR binding pockets

Posted by Peter Nollert on February 18, 2012
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

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M2 muscarinic acetylcholine receptor structure

Posted by Peter Nollert on January 31, 2012
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

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Comparing Apples with Oranges: B&W

Posted by Peter Nollert on November 7, 2011

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

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More options than I thought: GPCR expression systems

Posted by Peter Nollert on November 7, 2011
When it comes to expression systems, insect cells have been the primary supplier of GPCR protein for crystallographic studies. Notable exceptions are:
1. Rhodopsins can be obtained from the retina of eyes from Squid or cows and alternatively be expressed in COS cells.
2. 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

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Webinar on GPCR Expression for Biophysical and Structural Studies

Posted by Peter Nollert on October 11, 2011

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

GPCR expression webinar. Methods & techniques for expression of GPCRs for structural biology & biophysical methods

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