Receptors Flashcards
Traits of ionotropic receptors? (4 traits)
Ligand gated ion channel is itself the receptor
Made from multiple interchangeable protein subunits
Very fast (<50ms) at switching on/off
All or nothing action
Traits of metabotropic receptors?
System contains a channel but receptor is actually a single protein (often a GPCR)
Generally monomeric
Slow at switching on/off (100ms to minutes)
Can amplify or dampen signals
What type of receptors are Cys-loop receptors?
What is their structure and function? (hint - TM)
Ionotropic
Neurotransmitter receptor that can be a homo- or hetero-pentamer
Each monomer within the assembly has 4 TM helices
What does Nicotinic acetylcholine receptor (nAchrR) do and how?
What sub-class of receptor is it?
Mediates voluntary movement in skeletal muscle
Nerve signal releases acetylcholine from synaptic vesicles which binds and opens a cation channel on postsynaptic membrane triggering membrane depolarisation and muscle contraction
After a few ms the channel closes and acetylcholine is released to quickly switch off signal
Cys-loop receptor
Channel structure of nAChR? (hint - gate, residues)
Vestibule is wider at first and then narrows at the gate
It then opens wider towards the end into another vestibule
Vestibule contains certain residues based on its selectivity e.g. electronegative residues for positive ions
Only about 1/3 of nAChR receptor sits in membrane. What is the structure of its hydrophilic domain?
Ligand binding and coupling to channel opening?
M2 helices line channel
Acetylcholine binds to 2 sites at the interfaces of each α subunit with other subunits; This induces conformational change
Cys-loop pushes innermost domains, pushing valine loop against M2 inner helix
This opens up the pore so ions can go through
How is gating achieved in nAChR?
Gating is achieved by constriction and dilation at the narrowest point; Hydrophobic residues sticking into prevent hydrated ions
When gate is closed, its only closed just enough so that hydrated cation can’t fit through
How is crystallisation of GluCl aided?
Using water-soluble antibody fragments bound to extramembraneous domain to increase probability of good water contacts
nAChR is selective for a variety of cations and is excitatory
- It binds acetylcholine
By contrast glutamate gated chloride (GluCl) channels are anion selective and inhibitory
- They bind glutamate
These receptors have same basic architecture
How do ligands bind and how does GluCl achieve different specificity?
Both have ‘box-like’ binding site where 2 subunits come together
C loop closes around the ligand
GluCl binding pocket has residues that bind Glutamate rather than acetylcholine
How does GluCl achieve its selectivity?
Helical dipoles of M2 form 5 electro positive pockets at base of pore, sequestering anions
There are also electropositive residues along the vestibule
How does nAChR achieve its selectivity?
Has an excess of negatively charged groups on the inner wells of both vestibules
Rings of negatively charged side-chains are also located at the ends of the pore-lining helices and on the helices forming the intracellular domain to concentrate cations near the entrances of the narrow pore
What is Picrotoxin and what does it allow us to do?
Picrotoxin is an open channel blocker than traps a channel in open conformation
Helps with imaging and study
What are the 2 cys-loop channel states?
ELIC - Basal state
GLIC - Active state
How does a channel go from ELIC to GLIC?
Twist of β-sandwiches moves β1-β2 loops down and tilts M2 and M3 away from the central axis
Twisting of the extracellular domains tilts the 2 helices to open the pore
What can be done with freeze trapping?
Freeze trap, spray acetylcholine and use cryoEM to image acetylcholine binding to ligand binding domains and how it initiates rotational movements in the α-subunits which are then communicated to the inner M2 pore helices
Freeze trapping after spraying with acetylcholine shows how the channel is opened up for hydrated cation passage by just enough for the hydrated ions to fir through
What are nanodiscs?
Patch of lipid with 2 scaffold protein belts surrounding the patches hydrophobic tails
Nanodiscs allow us to mimic the membrane so we can isolate proteins for imaging easily
What can we do with nanodiscs? (hint - states)
What did they reveal? (hint - 3rd something)
Trap proteins in different states and use nanodiscs to image them with cryo-EM
Reveals a 3rd conformation - Desensitised
What is a desensitised receptor?
3 traits
Allows for the switching off of signalling by relaxing the conformation so ions cant pass through (not fully closed)
Ligand (e.g. glycine) is still bound
Second gate closed further down compared to closed state
Even when glycine is bound, gate can close to prevent passage of ions; Regulation
5 traits of GPCRs
7 TM helices
Can detect a variety of different stimuli
Ligand binds causing structural change in receptor, exciting G protein
When G protein is stimulated it interacts with effector
Opportunities for amplification
- One ligand can cause stimulation of many G proteins
What is the G-protein cycle? (4 steps with some end results)
Agonist binds receptor, exciting receptor
Gα subunit then binds cytoplasmic surface of receptor and opens up
Open Gα can now exchange GDP for GTP and potentially dissociate from βγ subunit
Activated G protein subunits then interact with effector proteins
- α subunit may interact with effectors such as adenylyl cyclase to convert ATP to cAMP
- βγ may interact with effectors such as channels
How is conformational change induced in rhodopsin? (hint - photon)
What does this conformational change then induce?
What is the G protein called?
Absorption of light results in a conformational change
This change catalyses the displacement of GDP by GTP which promotes dissociation of the G-protein (called transducin)
How is amplification seen in rhodopsin?
What does dissociated α subunit in rhodopsin do?
End result? (hint - hyperpolarisation)
1 photon can activate >500 molecules of transducin
Regulates a phosphodiesterase (switches it off), causing a drop in cGMP and ion channel closure
- 1 photon can result in the closure of 1000 channels
Membrane becomes hyperpolarised, triggering neurotransmitter release
Rhodopsin topology?
Arrestin?
7 TM helices
N-terminus on extracellular side; C-terminus on cytoplasmic
Arrestin binds cytoplasmic side, switching off receptor
5 key structural features on rhodopsin uncovered by X-ray crystallography?
Loop over top of α helices on extracellular side acts as lid
Pivot on TM helix 6 for flexible helix movement
Tryptophan is near retinal chromophore, which is bound to Lysine
Retinal is positively charged in resting state, so there are some Glutamate counterions
Helix 6 moves on cytoplasmic side
What residue is attached to retinal and why? (hint - light absorption)
Why not tryptophan?
Retinal is attached to Lys296 in post-translational modification as it’s a much better absorber of visible light
Tryptophan can absorb invisible light but doesn’t have a large extinction coefficient
How does opsin become rhodopsin?
How/where is retinal attached? (hint - cis)
Once retinal is attached to opsin
Retinal is attached with 11-cis conformation (bent) in resting/dark state
How does retinal isomerisation occur? (hint - trans)
- Protein state? (hint - Meta)
What happens then? (hint - proton, bleaches)
How is system reset?
Photon hits retinal and it becomes all-trans and straightens out, putting strain on protein
Protein is now in Meta II state
Retinal loses a proton and is removed and processed into retinol by retinal dehydrogenase; Bleaches protein
Downstream recycling resets system by putting new retinal back into protein
How is the retinal binding pocket so tightly packed? (hint - ring, lid)
Residues positioned closely around it make it a very snug fit
Tryp265 ring stacked against cyclohexane ring of retinal; Very close interaction
Lids encapsulate binding pocket to protect from outside of cell
How does straightening of retinal put strain on protein?
Residues very close to retinal causing Van der Waals to be in close contact
Retinal isomerisation puts strain on the residues around it as it straightens out
- Light energy converted to mechanical force, causing protein to change structure
How long for Metarhodopsin II (Meta II or R*) state to be formed?
What base is deprotonated to form this state?
What does this state result in?
Formed within 1ms
Schiff base with retinal is deprotonated, causing a significant structural change in opsin
This state results in G protein (transducin) activation
What receptor (and state) is analogous to Meta II state?
What can we do with this?
Analogous to ligand-bound state of β2-Adrenergic receptor
These receptors can be used as models for one another
In structural studies, what are the 2 ways we can look at what happens when the strain is relaxed and how that activates transducin? (hint - remove retinal, mutants)
Remove retinal and add peptide fragment of transducin Gα subunit; Evidence that conformation we end up with represents Meta-II active state
Use mutants with constitutive activity (trap membrane protein in active state) e.g. E113Q
What structural feature is key in ground/dark state of rhodopsin?
Lid covering and blocking binding site for retinal
In the dark state, rhodopsin has no cavity for what? (hint - steric clash)
In the excited/ligand-bound state how does this change? (hint - extension and pushed out)
Why do we need it to bind?
No cavity for Gα subunit to bound as TM helices cause steric clash with any potential Gα C terminus so it can’t bind
Ligand-induced conformational change causes TM helices 5 to extend and 6 and 7 to be pushed out on intracellular side, opening a space for Gα subunit and allowing it to bind
Gα subunit needs to bind so it can be opened and exchange GDP for GTP
What is an ionic lock and how is it different in inactive and active state?
What is this effectively?
Arg135 (+ve) on TM helix 3 bound to glutamates (-ve) on TM helix 6 through salt bridge and H-bonding networks
- This forms an ionic lock which stabilises inactive state
Movement of TM6 breaks the lock and Arg135 now interacts with Gα and is stabilised by tyrosines, another lock
Effectively this is a molecular switch
What features are similar between rhodopsin and other members of its family? (3 things)
Spatial arrangement of TM helices
Some water molecule locations are conserved
- May have important functional role in structure and changes in structure
Ionic lock is conserved
How does ligand binding site of rhodopsin compare with other receptors in this family?
- Similarities and difference?
Very similar location
Same strain and pushing of TM 6 for G protein activation
Binding residues in each binding site are slightly different
Compare rhodopsin and β2-adrenergic receptor structures (hint - extramembraneous)
Similar overall folds
Similar binding pockets
Extramembraneous loops quite different; Confer different ligand specificities
β2AR is similar to rhodopsin, but is involved in different signalling pathways
How is it different?
- Activation
- Downstream signalling
- Deactivation and internalisation?
β2AR binds ligand to be activated instead of light
Gα switches off adenylate cyclase; Drop in cAMP
Channel stimulated by cAMP closes and there is hyperpolarisation
β2AR can be deactivated/desensitised through phosphorylation of β2AR and arrestin recognising phosphorylated state
β2AR-arrestin complex is then internalised and recycled/degraded
There are different types of signal coming from β2AR, with varying levels of response
What does this depend on and give the different responses? (5 response levels)
Depends on the ligand bound
Exists at low level basal activity (not fully off)
Full agonist stimulates full response
Partial agonist stimulates reduced response
Antagonists switch off receptor and lock it at basal activity
Inverse agonists reduce activity below basal level
What was used to get crystal structure of β2AR? (hint - chimera)
How was inactive state stabilised?
Chimera molecule of β2AR and phage T5 lysozyme (T4L) fragment
This stabilises the structure and gives extra soluble surface for forming crystal contacts
Inverse agonist bound to stabilise inactive state for crystallisation
Through overlapping and comparing rhodopsin and β2AR structures, what differences were uncovered? (hint - lid, specificity)
Different arrangement makes for very different binding
β2AR has different residues around ligand to confer different specificity
Also has no cap/lid; Passageway allowing ligand to soak in
β2AR binding site is in roughly the same place as Rhodopsin’s for retinal
What contributes to a tight binding site in β2AR?
- Use carazolol as ligand example (polar tail, hydrophobic ring)
Binding site has polar residues which interact with polar tail of ligand
Aromatic/hydrophobic residues interact with ring structure in ligand
As carazolol has very low Kd and slow off-rate, it has tight binding
What structure makes this binding even tighter? (hint - sammidge)
Hydrophobic sandwich
2 hydrophobic layers enclose and ‘sandwich’ the hydrophobic parts of the ligand, making it tighter and less likely to release
How are the receptor interactions different between full, partial agonists and antagonists?
Full - All necessary reactions with surrounding residues to incur
Partial - Only has some of the interactions, so structural changes give a weakened response
Antagonists - Bind additional residues, giving different structural changes which give no response increase
How is ionic lock broken in β2AR? (hint - rhodopsin)
Similar to rhodopsin mechanism when full agonist binds
TM5 is extended and TM 6 is pushed out allowing for G protein to bind
Different residue involved in stabilising each state of ionic lock but same concept
How can we crystallise β2AR in complex with a G protein?
T4L fusion and detergent micelles can be used to crystallise active receptor
What are the differences between inactive and active state of β2AR? (hint - movement, hinge)
Main difference between active state with bound G protein and inactive state is large movement of TM 6 and TM 5 extending
In inactive state Gα is hinged around binding site so GDP is not exposed and can’t be exchanged
How does Gα open and what does this allow?
Gα opens when you get conformational change of TM 5 and 6 in the receptor
These helices form a hinge over binding site so this allows it to bind and then open
Then allows Gα to open and expose nucleotide binding site and exchange GDP for GTP
What is an analgesic?
What receptor do many analgesics bind?
What trait isn’t ideal?
Medications that relieve pain e.g. by reducing inflammation
Bind α2A-Adrenergic Receptor (α2AAR) as its activation has pain-relieving effects
Many analgesic are strongly sedating, likely binding to other receptors
How did they test many molecules to assess their binding to α2A-Adrenergic Receptor (α2AAR)? (hint - DOCK)
What does the score depend on?
Did a computational screen using ZINC15 database to test molecules and their binding, giving each molecule a score using DOCK software
Score depends on interactions between ligand and binding site; Van der Waal energy, electrostatic, ligand desolvation energy
What traits did new agonists found for (α2AAR) via docking tests show?
Low nM to low μM binding constants
- Need even tighter binding
All have 6C rings as well as linker groups between rings
- Similar features
What did docking derived agonist show that other drugs didn’t?
Preferentially activated a narrow range of G protein subtypes,
Contrasts with drugs, like dexmedetomidine and brimonidine, that activate a much broader set of G proteins which likely causes sedative effect
What was used to optimise initial docking hits? (hint - cryo, EC50)
CryoEM structure determinisations provided templates for optimisation of the initial docking hits
Used it to make tweaks to molecules to maker tighter binding and lower EC50
