Post on 02-Jul-2015
description
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).