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, GebhardSchertler, Christopher Tate

Presented by Lucas Man

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

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

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?

⇌

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

Methods

1. Receptor expression

2. Receptor purification and crystallization

3. X-ray crystallography and analysis

Picture sources: U of Miami, West Kentucky U

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

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

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

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

Results

• All 4 agonists bind in the catecholamine pocket

Figure 1 – Structure of β1-adrenergic receptor bound to agonists

Results

Figure 4 – Differences in the ligand-binding pocket between antagonist- and agonist-bound β1-adrenergic receptor

Orange: IsoprenalineGrey: Cyanopindolol

• 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

Results

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

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

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

References

• Warne, T. et al. “Structural basis for agonist and partial agonist action on a β1-adrenergic receptor”. Nature. Vol469, 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).