The structural basis for agonist and partial agonist action on the β1-adrenergic receptor was investigated. X-ray crystallography revealed that full agonists stabilized the ligand-binding pocket and induced conformational changes that facilitated the transition to the active receptor state. Partial agonists were less effective at stabilizing the pocket and inducing these changes, explaining their intermediate efficacy. Antagonists functioned as very weak partial agonists by providing little stabilization or conformational changes.

1. The structural basis for agonist and
partial agonist action on a β1-
adrenergic receptor
Tony Warne, Rouslan Moukhametzianov,
Jillian Baker, Rony Nehme, Patricia
Edwards, Andrew Leslie, Gebhard
Schertler, Christopher Tate
Presented by Lucas Man

2. Introduction
• Adrenergic receptors and
other G-protein-coupled
receptors play important
roles in biosignaling
▫ β1-adrenergic receptors in
the heart
▫ Many drugs are synthetic
ligands
• There is a range of ligand-
binding effects; partial
agonists
▫ The mechanism of this is
not well understood

3. Some biochemistry
• Protein receptors usually
have 2 structurally
different states:
▫ Inactive R state
▫ Active R* state
• R* state couples with G-
protein, activates
cascade
• At equilibrium
Source: NCBI

4. Some biochemistry
• Standard conditions: R state preferred
• Agonist binding stabilizes R* state: R* state preferred
• Antagonists block agonists from binding
⇌
• Partial agonists?
▫ How are intermediate cellular responses produced?

5. Hypothesis
• Receptors are either in R or R* state
▫ No evidence for intermediate state with reduced
function
• Hypothesis:
▫ Partial agonists stabilize the R* state, but to a
lesser extent than full agonists
▫ Equilibrium shifted towards R*, but to a lesser
extent

6. Methods
1. Receptor expression
2. Receptor purification and crystallization
3. X-ray crystallography and analysis
Picture sources: U of
Miami, West Kentucky U

7. 1. Receptor expression
• Recombinant Baculovirus construct
▫ Gene for turkey thermostabilized β1 AR-m23 with His
tag spliced in
• Infection of insect cells
• β1 AR-m23 produced in infected cells
Source:
NCBI

8. 2. Receptor purification and
crystallization
• Centrifuge cells to separate proteins
• Use Immobilized Metal Ion Affinity Chromatography
(IMAC) to isolate receptor
▫ Nickel column
▫ His tag binds to Ni
Picture sources:
Wikimedia

9. 2. Protein purification and crystallization
• Separate isolated receptor proteins into 5 different
solutions:
▫ R-Isoprenaline (full agonist)
▫ R,R-carmoterol (full agonist)
▫ R-salbutamol/albuterol (partial agonist)
▫ R-dobutamine (partial agonist)
▫ Cyanopindolol (antagonist)
• Hanging drop, vapor diffusion crystallization
Source: Wikipedia (Protein crystallization) Source: NASA

10. 3. X-ray crystallography and analysis
• Electron clouds diffracts x-ray beams
• Diffraction pattern can be used to create an electron
density map
• Use computer software to fit known amino acid sequence
into the electron density map
Picture sources:
U of Arizona, Rice U

11. Results
• All 4 agonists bind in the catecholamine pocket
Figure 1 – Structure of
β1-adrenergic receptor
bound to agonists

12. Results
Figure 4 – Differences
in the ligand-binding
pocket between
antagonist- and agonist-
bound β1-adrenergic
receptor
Orange: Isoprenaline
Grey: Cyanopindolol

13. Results
• What does this all mean?
• Full agonists formed more hydrogen bonds to receptor
helices than partial agonists
▫ Stabilization of ligand-binding pocket
• Full agonists induced key conformational changes in
certain amino acid residues
▫ Ser212, Ser215
• Strengthen H5-H6 interface, weaken H4-H5 interface
▫ Facilitate movement of helixes to R* conformation

14. Results
• 3 major determinants of ligand efficacy:
1. Ser212 conformational change
2. Ser215 conformational change
3. Stabilization of contracted ligand-binding pocket
• Full agonists achieved all 3
• Partial agonists failed at #2 and may be less
successful at #3
▫ Supports hypothesis
• Antagonist functioned as very weak partial agonist

15. Summary
• Agonist-receptor binding
▫ Stabilizes binding pocket
▫ Ease transition to R* state
• Partial agonist-receptor binding
▫ Less stabilization of binding pocket
▫ Less conformational changes
• Antagonist-receptor binding
▫ Little to no stabilization of binding pocket
▫ Little to no conformational changes

16. Importance
• Still a lot of speculation
▫ 2 independent structures found for dobutamine-bound
receptor
▫ Differences between thermostabilized and natural β1-
adrenergic receptors
▫ Only initial binding state observed
• G-protein-coupled receptors have highly conserved
amino acid sequences and structural similarities
• What applies to β1-adrenergic receptor probably
applies to other G-protein coupled receptors as well

17. References
• Warne, T. et al. “Structural basis for agonist and partial
agonist action on a β1-adrenergic receptor”. Nature. Vol
469, pp 241-244 (13 Jan 2011).
• Warne, T. et al. “Development and crystallization of a
minimal thermostabilised G-protein-coupled receptor”.
Protein Expr. Purif. Vol 65, pp 204-213 (2009).