PSI Structural Biology Knowledgebase

PSI | Structural Biology Knowledgebase
Header Icons
E-Collection

Related Articles
Drug Discovery: Solving the Structure of an Anti-hypertension Drug Target
July 2015
Retrospective: 7,000 Structures Closer to Understanding Biology
July 2015
Families in Gene Neighborhoods
June 2015
Channels and Transporters: BEST in Show
April 2015
Channels and Transporters: Reorienting a Peptide in the Pocket
April 2015
Ryanodine Receptor
April 2015
Protein Folding and Misfolding: It's the Journey, Not the Destination
March 2015
Protein Folding and Misfolding: Refolding in Membrane Mimetic
March 2015
Nuclear Pore Complex: A Flexible Transporter
February 2015
Nuclear Pore Complex: Higher Resolution of Macromolecules
February 2015
Nuclear Pore Complex: Integrative Approach to Probe Nup133
February 2015
Piecing Together the Nuclear Pore Complex
February 2015
Mitochondrion: Flipping for UCP2
December 2014
Transmembrane Spans
December 2014
Glucagon Receptor
April 2014
Membrane Proteome: A Cap on Transport
April 2014
Membrane Proteome: Microcrystals Yield Big Data
April 2014
Membrane Proteome: Pumping Out Heavy Metal
April 2014
Design and Discovery: Virtual Drug Screening
January 2014
G Proteins and Cancer
November 2013
Drug Discovery: Antidepressant Potential of 6-NQ SERT Inhibitors
October 2013
Drug Discovery: Modeling NET Interactions
October 2013
Microbiome: Solid-State NMR, Crystallized
September 2013
CAAX Endoproteases
August 2013
Membrane Proteome: A Funnel-like Viroporin
August 2013
Membrane Proteome: GPCR Substrate Recognition and Functional Selectivity
August 2013
Membrane Proteome: Making DNA Nanotubes for NMR Structure Determination
August 2013
Membrane Proteome: Unveiling the Human α-helical Membrane Proteome
August 2013
Cell-Cell Interaction: Magic Structure from Microcrystals
March 2013
Cell-Cell Interaction: Nanoparticles in Cell Camouflage
March 2013
Membrane Proteome: Capturing Multiple Conformations
December 2012
Membrane Proteome: Soft Sampling
December 2012
Membrane Proteome: Sphingolipid Synthesis Selectivity
December 2012
Membrane Proteome: Tuning Membrane Protein Expression
December 2012
Cytochrome Oxidase
November 2012
Membrane Proteome: Building a Carrier
November 2012
Membrane Proteome: Every Protein Has Its Tag
November 2012
Membrane Proteome: Specific vs. Non-specific weak interactions
November 2012
Membrane Proteome: The ABCs of Transport
November 2012
Bacterial Phosphotransferase System
October 2012
Insert Here
October 2012
Solute Channels
September 2012
To structure, faster
August 2012
Pocket changes
July 2012
Predictive protein origami
July 2012
G Protein-Coupled Receptors
May 2012
Twist to open
March 2012
Anchoring's the way
February 2012
Overexpressed problems
February 2012
Gentle membrane protein extraction
January 2012
Docking and rolling
October 2011
A fragmented approach to membrane protein structures
September 2011
Raising a glass to GLIC
August 2011
Sugar transport
June 2011
A2A Adenosine Receptor
May 2011
TrkH Potassium Ion Transporter
April 2011
Subtly different
March 2011
A new amphiphile for crystallizing membrane proteins
January 2011
CXCR4
January 2011
Guard cells pick up the SLAC
December 2010
ABA receptor diversity
November 2010
COX inhibition: Naproxen by proxy
November 2010
Zinc Transporter ZntB
July 2010
Formate transporter or channel?
March 2010
Tips for crystallizing membrane proteins in lipidic mesophases
February 2010
Urea transporter
February 2010
Five good reasons to use single protein production for membrane proteins
January 2010
Membrane proteins spotted in their native habitat
January 2010
Spot the pore
January 2010
Get3 into the groove
October 2009
GPCR subunits: Separate but not equal
September 2009
GPCR modeling: any good?
August 2009
Surviving in an acid environment
August 2009
Tips for crystallizing membrane proteins
June 2009
You look familiar: the Type VI secretion system
June 2009
Bacterial Leucine Transporter, LeuT
May 2009
Aquaglyceroporin
March 2009
Death clusters
March 2009
Protein nanopores
March 2009
Transporter mechanism in sight
February 2009
A pocket guide to GPCRs
December 2008
Tuning membrane protein overexpression
October 2008
Blocking AmtB
September 2008

Research Themes Membrane proteins

A pocket guide to GPCRs

PSI-SGKB [doi:10.1038/fa_psisgkb.2008.16]
Featured Article - December 2008
Short description: The structure of the G-protein-coupled receptor adenosine receptor A2A reveals differences between its ligand-binding site and those of other family members, and begins to explain this family's diversity.Science, doi:10.1126/science.1164772

Structure of the human adenosine A2a receptor bound with the potential Parkinson's drug ZM241385 (blue). Four disulfide bridges (yellow) form a complex network to shape the extracellular loops (green) and binding site region. Image courtesy of the Stevens Laboratory, The Scripps Research Institute. (PDB 3EML)

G-protein-coupled receptors (GPCRs) are implicated in numerous diseases and are the target of many drug treatments. Obtaining structures of these receptors has been a challenge, and is still proving to be difficult. Until recently, of the almost 1,000 proteins in this family, only two crystallographic structures had been solved.

Now, a third family member has been added. Jaakola et al., part of the NIH Roadmap center JCIMPT and PSI center ATCG3D, in collaboration with colleagues from the Leiden/Amsterdam Center for Drug Research, have elucidated the structure of the human adenosine A2A receptor.

The adenosine A2A receptor is a class A GPCR, and is important for neurotransmission, coronary blood flow and respiration. It is blocked by caffeine, and is the subject of much research after epidemiological evidence suggested that coffee drinkers have a lower risk of Parkinson's disease. Selective compounds are likely to be useful for the treatment of pain, Parkinson's disease, Huntington's disease and asthma.

Crystals of GPCRs, in general, are difficult to obtain because of the receptors' flexibility and because of their structural heterogeneity. The structures previously solved, rhodopsin and the human β2 adrenergic receptors, are among the most studied GPCRs. The adenosine A2A receptor, similar to the β2-adrenergic receptor, is rapidly denatured upon concentration without the presence of an inverse agonist or antagonist.

The strategy

To solve the problem of the adenosine A2A receptor's instability and to increase the chances of obtaining crystals, Jaakola et al. used a T4L fusion strategy. For this, they replaced the third cytoplasmic loop (Leu209–Ala221) of the receptor with lysozyme from T4 bacteriophage, deleted the C-terminal tail, and conducted all structural studies in a cholesterol-enriched lipidic cubic phase (LCP). Multiple technologies had to be developed to successfully observe, extract and collect X-ray diffraction data on the delicate and tiny crystalline samples.

The resulting receptor was also stabilized during purification by adding sodium chloride and a saturating concentration of a non-specific adenosine receptor antagonist, which was swapped for its specific antagonist ZM241385 at the last step. In addition, cholesteryl hemisuccinate was present throughout purification. Comparison of the binding properties of the fusion construct with the wild-type receptor confirmed that the antagonist binds with similar affinity to both receptors.

Using the T4L fusion protein, the team obtained the crystal structure of the human adenosine A2A receptor in complex with a high-affinity A2A-selective antagonist ZM241385 at 2.6 Å resolution. It reveals three features that are distinct from those of previously reported GPCR structures and that explain the selectivity of this receptor.

Family differences

The first discrepancy is that the antagonist ZM241385 binds to A2A in an extended conformation perpendicular to the plane of the membrane. This is different from predictions from earlier modeling studies, based on the structures of known GPCR structures, that docked ZM241835 in a binding site that was like that of β2-adrenergic receptors and rhodopsin.

The subtle differences in helical positions and orientations relative to rhodopsin and the β2-adrenergic receptors created this new antagonist-binding cavity, and show an important role for extracellular loops and helical core in ligand recognition.

The second difference is in the extracellular loop organization. That of the adenosine A2A receptor is mainly a spatially constrained random coil, whereas rhodopsin and β-adrenergic receptors have some secondary structure elements such as a β-sheet and α-helix.

The third discrepancy is that the antagonist for adenosine A2A restricts the movement of a tryptophan residue important for activating all three GPCR receptors. This tryptophan is thought to act as a 'toggle switch', and Jaakola et al. suggest that ZM241385 prevents structural rearrangements needed for activation of adenosine A2A, thus locking it in the inactive/resting state.

Overall, this structure suggests that there is no general, family-conserved receptor-binding pocket for GPCRs. Instead, the pocket varies in position and orientation, which would give rise to diverse receptors and enhance ligand selectivity.

Maria Hodges

References

  1. Veli-Pekka Jaakola, Mark T. Griffith, Michael A. Hanson, Vadim Cherezov, Ellen Y. T. Chien et al. The 2.6 Å crystal structure of a human A2A adenosine receptor bound to an antagonist.
    Science (2 October 2008). doi:10.1126/science.1164772

Structural Biology Knowledgebase ISSN: 1758-1338
Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health