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.
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
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!
June 23, 2011
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,
March 28, 2011
Thanks to all readers of this blog for making me aware of the recently published structure of the agonist UK-432097 - bound Adenosine A2A crystallographic structure: (3QAK). Congratulations (sic!) to the Stevens group!
Xu F, Wu H, Katritch V, Han GW, Jacobson KA, Gao ZG, Cherezov V, & Stevens RC (2011).Structure of an Agonist-Bound Human A2A Adenosine Receptor. Science PMID: 21393508
This new GPCR structure shows (again) how agonist binding triggers internal structural rearrangements that lead to activiation, reminiscent of rhodopsin activation. Even if you're only marginally interested in GPCR structural biology, I think you'd enjoy experiencing these interactive and fun 'movies' that illustrate the conformational shifts that are triggered by the binding event.
Interestingly, this A2A receptor agonist UK-432097 induces additional conformation changes that the authors deem specific and hence call this particular liagand a 'conformationally selective agonist' that stabilizes the receptor activated state.
There's a pattern.
January 22, 2011
Wow, the new year is off to a great start in GPCR structural biology:
Rasmussen SG, Choi HJ, Fung JJ, Pardon E, Casarosa P, Chae PS, Devree BT, Rosenbaum DM, Thian FS, Kobilka TS, Schnapp A, Konetzki I, Sunahara RK, Gellman SH, Pautsch A, Steyaert J, Weis WI, & Kobilka BK (2011). Structure of a nanobody-stabilized active state of the β(2) adrenoceptor. Nature, 469 (7329), 175-80 PMID: 21228869
Rosenbaum DM, Zhang C, Lyons JA, Holl R, Aragao D, Arlow DH, Rasmussen SG, Choi HJ, Devree BT, Sunahara RK, Chae PS, Gellman SH, Dror RO, Shaw DE, Weis WI, Caffrey M, Gmeiner P, & Kobilka BK (2011). Structure and function of an irreversible agonist-β(2) adrenoceptor complex. Nature, 469 (7329), 236-40 PMID: 21228876
Warne T, Moukhametzianov R, Baker JG, Nehmé R, Edwards PC, Leslie AG, Schertler GF, & Tate CG (2011). The structural basis for agonist and partial agonist action on a β(1)-adrenergic receptor. Nature, 469 (7329), 241-4 PMID: 21228877
This will take some time to digest,
December 30, 2010
The 2008 'pocket guide to GPCRs' - structure determination of GPCRs that is, - still serves well. Just after the Adenosine A2A receptor structure was published in October 2008, this featured article in Nature Structural Biology Knowledgebase laid out the following strategy for successful determination of crystallographic GPCR structures:
Increase compactness & stability: shorten third cytoplasmic loop; identify stabilizing mutations, such as C-terminal deletion
Adjust purification regime: add cholesterol hemisuccinate throughout purification
Increase crystallizability: replace long loop section of third intracellular loop by T4 lysozyme fusion
Stabilize protein during crystallization: employ LCP crystallization methodology use monoolein with added cholesterol; co-crystallize with ligand
In 2008 this was a just a trend (N=3), now at the end of 2010 with 2 more GPCR structures in our pocket, both of which follow these guidelines, this pocket guide is starting to look like a rule book.
All the best,
Reference: Hodges, M. (2008). A pocket guide to GPCRs PSI Structural Genomics Knowledgebase DOI: 10.1038/fa_psisgkb.2008.16
December 24, 2010
What are readers of this GPCR blog most interested in? Below is the list of the top 10 blog posts that were requested often during this year. If this is close to what you’re interested in, you may want to subscribe via RSS.
So, here are the top ten blog posts:
No surprise in the choice of topic: structure, structure, structure.
This should set the theme for 2011,
December 18, 2010
This year we’ve seen 2 new X-ray GPCR structures published, and the PDB now shows 27 entries (list here).
Let’s put this progress into a historical perspective: The first protein structure was published in 1960 (Kendrew et al.), the first membrane protein structure in 1985 (Deisenhofer, et.al) and the first GPCR structure appeared in the PDB in 2000 (Palczewski et al.). As of today the Protein Data Bank lists a staggering 69,967 entries (soluble and membrane protein structures, with a variety to techniques used), of these about 719 are membrane protein structures, of which 262 are unique.
What does that tell us for GPCR structures in 2011? One could reasonably expect to see two or three new unique GPCR structures in 2011 and maybe an additional 10 GPCR structures that are related to already determined ‘base structures’ (for instance the advertised antagonist bound b2AR structure). This estimate is a continuation of a trend that was first described by Richard Dickerson in 1978 for soluble proteins and then applied to membrane proteins by Stephen White (see here for Stephen’s fantastic Membrane Proteins of known 3D Structure site).
The numbers to extend this Dickerson-White law to GPCRs by fitting m = exp(by) are still somewhat shaky for GPCRs, but if the exponential trend holds true we could reasonably expect more than 2 and less than 3 unique GPCR structures in 2011 (one of them: S1P receptor). Another way to look at GPCR structural biology is that, compared to soluble proteins, GPCR structure determination is back in the mid nineteen seventies.
We’ve got a long way to go,
First Protein Structure:
KENDREW JC, DICKERSON RE, STRANDBERG BE, HART RG, DAVIES DR, PHILLIPS DC, & SHORE VC (1960). Structure of myoglobin: A three-dimensional Fourier synthesis at 2 A. resolution. Nature, 185 (4711), 422-7 PMID: 18990802
First Membrane Protein Structure:
Deisenhofer J., Epp O., Miki K., Huber R., and Michel H. 1985. Structure of the protein subunits in the photosynthetic reaction centre of Rhodospeudomonas viridis at 3 Å resolution. Nature 318:618-624.
First X-ray crystallographic structure of a G-protein coupled receptor:
Palczewski, K. (2000). Crystal Structure of Rhodopsin: A G Protein-Coupled Receptor Science, 289 (5480), 739-745 DOI: 10.1126/science.289.5480.739
December 11, 2010
I was recently asked "which of the published GPCR structures…are most relevant to drug discovery?"
Like many simple sounding questions there's no way to give a straight answer. Why is that? I think that the relevance of a particular GPCR structure is in the eye of the beholder. Let's look at the GPCR structures out there now and start with the seemingly simplest one to give a poor ranking: rhodopsin. One could say that the relevance of this structure has decreased for those molecular modelers that require starting coordinates to build accurate GPCR models of other receptors. The non-rhodopsin structures should serve as better starting points. On the other hand, since there is structural data available for rhodopsin in different conformational substates (photoactivated and ground states, with and without retinal ligand, nicely defined water molecules) there is currently no other GPCR that's better understood in terms of detailed function. The latter is supported by a wealth of spectroscopic and biochemical work that paints the most detailed picture (movie!) of receptor function.
What about Dopamine D3, beta 1 / 2 Adrenergic receptors and the Adenosine receptor? Here one could argue that for those researchers who want to discover new drugs, the structures of these receptors may not be that helpful since there's ample evidence (i.e. drugs out in the marketplace) that it is fundamentally possible to develop compounds that interact with these receptors in desired ways. Hence no need for structural insight. I'm playing devils advocate here, because having access to accurate structural models and a structure-producing method does increase the efficiency for structure guided drug discovery efforts a lot. And it may even provide fundamentally new starting points, for instance by applying fragment-based crystallographic screening.
What about the CXCR4 receptor then? Certainly a highly relevant target (HIV) - and my personal favorite; however, if you want to make an impact on as many peoples' health as possible, you may want to focus on cardiovascular diseases and neurological disorders instead.
So, if you have a research agenda, you better know which GPCR structure is closest to you. For those asking which GPCR structure is the most relevant to drug discovery I say 'those ones we haven't see yet'.
Does that make sense?
December 4, 2010
Since GPCRs are transmembrane proteins they require to be removed from the membrane for their isolation, chromatographic purification and growth of crystals for X-ray structural analysis. This solublization process is facilitated by amphiphilic reagents that provide a microenvironment that is compatible with aqueous solutions and maintains the proper structural integrity of the membrane protein as individual particles. The list of the most useful amphile reagents, detergents, is long for membrane proteins in general, but short for GPCRs. In total, 49 unique detergents (including detergent mixtures) have been used to grown membrane protein crystals (Newstead et al., 2008). Interestingly, it turns out that with only 8 detergents, 80 % of membrane protein crystals can be grown. Below is the short table of those detergents that have been applied to the purification and crystallization of GPCRs.
Detergents used for extraction & solubilization of GPCRs for 3D crystallization:
- b1Adrenergic Receptor
- Bovine Rhodopsin
- b2Adrenergic Receptor
- Adenosine A2A Receptor
- Dopamine D3 Receptor
- Nonyl beta glucoside / heptantriol
- Dodecyl maltoside / cholesterol hemisuccinate
- Dodecyl maltoside / cholesterol hemisuccinate
- Dodecyl maltoside / cholesterol hemisuccinate
- Dodecyl maltoside / cholesterol hemisuccinate
Of course, for the G-protein coupled receptors that were crystallized in a lipidic cubic phase matrix, monoolein and cholesterol played a major role. Nonetheless, the extraction from the original membrane and purification requires one to select a particular detergent. Hence, for GPCRs the list of tried-and-proven detergents offers only three choices: 1. octylthioglucoside, 2. nonylbetaglucoside (with heptanetriol) or 3. dodecyl maltoside (with cholesterol hemisuccinate). Fortunately this collection has just grown dramatically. In a recent Nature Methods paper that I review in this blog post (New Detergent Class for Membrane Proteins) over at the Protein Crystallization Hits blog, a new class of detergents, called maltose-neopentyl glycol amphiphiles (MNGs) is introduced.
'Hidden' in the body of the paper is this gem of good news: one of the MNGs has produced crystals of b2AR-T4L that could not be grown by using the standard DDM (dodecylmaltoside) detergent. This bodes well for experimentalists that are stuck with the current narrow choice of detergents. And this is also good news for everybody who's been waiting for a structure of the agonist-bound beta 2 adrenergic receptor, the latter of which is advertised in the MNG methods paper (Chae et al. , 2010) to become avaliable at 3.5A resolution.
Newstead, S., Ferrandon, S., & Iwata, S. (2008). Rationalizing α-helical membrane protein crystallization Protein Science, 17 (3), 466-472 DOI: 10.1110/ps.073263108
Chae PS, Rasmussen SG, Rana RR, Gotfryd K, Chandra R, Goren MA, Kruse AC, Nurva S, Loland CJ, Pierre Y, Drew D, Popot JL, Picot D, Fox BG, Guan L, Gether U, Byrne B, Kobilka B, & Gellman SH (2010). Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nature methods, 7 (12), 1003-8 PMID: 21037590