Receptors

Question 1

Traits of ionotropic receptors? (4 traits)

Answer 1

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

Question 2

Traits of metabotropic receptors?

Answer 2

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

Question 3

What type of receptors are Cys-loop receptors?
What is their structure and function? (hint - TM)

Answer 3

Ionotropic

Neurotransmitter receptor that can be a homo- or hetero-pentamer
Each monomer within the assembly has 4 TM helices

Question 4

What does Nicotinic acetylcholine receptor (nAchrR) do and how?
What sub-class of receptor is it?

Answer 4

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

Question 5

Channel structure of nAChR? (hint - gate, residues)

Answer 5

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

Question 6

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?

Answer 6

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

Question 7

How is gating achieved in nAChR?

Answer 7

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

Question 8

How is crystallisation of GluCl aided?

Answer 8

Using water-soluble antibody fragments bound to extramembraneous domain to increase probability of good water contacts

Question 9

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?

Answer 9

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

Question 10

How does GluCl achieve its selectivity?

Answer 10

Helical dipoles of M2 form 5 electro positive pockets at base of pore, sequestering anions
There are also electropositive residues along the vestibule

Question 11

How does nAChR achieve its selectivity?

Answer 11

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

Question 12

What is Picrotoxin and what does it allow us to do?

Answer 12

Picrotoxin is an open channel blocker than traps a channel in open conformation
Helps with imaging and study

Question 13

What are the 2 cys-loop channel states?

Answer 13

ELIC - Basal state
GLIC - Active state

Question 14

How does a channel go from ELIC to GLIC?

Answer 14

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

Question 15

What can be done with freeze trapping?

Answer 15

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

Question 16

What are nanodiscs?

Answer 16

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

Question 17

What can we do with nanodiscs? (hint - states)
What did they reveal? (hint - 3rd something)

Answer 17

Trap proteins in different states and use nanodiscs to image them with cryo-EM

Reveals a 3rd conformation - Desensitised

Question 18

What is a desensitised receptor?
3 traits

Answer 18

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

Question 18

5 traits of GPCRs

Answer 18

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

Question 19

What is the G-protein cycle? (4 steps with some end results)

Answer 19

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

Question 20

How is conformational change induced in rhodopsin? (hint - photon)
What does this conformational change then induce?
What is the G protein called?

Answer 20

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)

Question 21

How is amplification seen in rhodopsin?
What does dissociated α subunit in rhodopsin do?
End result? (hint - hyperpolarisation)

Answer 21

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

Question 22

Rhodopsin topology?
Arrestin?

Answer 22

7 TM helices

N-terminus on extracellular side; C-terminus on cytoplasmic

Arrestin binds cytoplasmic side, switching off receptor

Question 23

5 key structural features on rhodopsin uncovered by X-ray crystallography?

Answer 23

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

Question 24

What residue is attached to retinal and why? (hint - light absorption) Why not tryptophan?

Answer 24

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

Question 25

How does opsin become rhodopsin? How/where is retinal attached? (hint - cis)

Answer 25

Once retinal is attached to opsin Retinal is attached with 11-cis conformation (bent) in resting/dark state

Question 26

How does retinal isomerisation occur? (hint - trans) - Protein state? (hint - Meta) What happens then? (hint - proton, bleaches) How is system reset?

Answer 26

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

Question 27

How is the retinal binding pocket so tightly packed? (hint - ring, lid)

Answer 27

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

Question 28

How does straightening of retinal put strain on protein?

Answer 28

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

Question 29

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?

Answer 29

Formed within 1ms Schiff base with retinal is deprotonated, causing a significant structural change in opsin This state results in G protein (transducin) activation

Question 30

What receptor (and state) is analogous to Meta II state? What can we do with this?

Answer 30

Analogous to ligand-bound state of β2-Adrenergic receptor These receptors can be used as models for one another

Question 31

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)

Answer 31

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

Question 32

What structural feature is key in ground/dark state of rhodopsin?

Answer 32

Lid covering and blocking binding site for retinal

Question 33

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?

Answer 33

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

Question 34

What is an ionic lock and how is it different in inactive and active state? What is this effectively?

Answer 34

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

Question 35

What features are similar between rhodopsin and other members of its family? (3 things)

Answer 35

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

Question 36

How does ligand binding site of rhodopsin compare with other receptors in this family? - Similarities and difference?

Answer 36

Very similar location Same strain and pushing of TM 6 for G protein activation Binding residues in each binding site are slightly different

Question 37

Compare rhodopsin and β2-adrenergic receptor structures (hint - extramembraneous)

Answer 37

Similar overall folds Similar binding pockets Extramembraneous loops quite different; Confer different ligand specificities

Question 38

β2AR is similar to rhodopsin, but is involved in different signalling pathways How is it different? - Activation - Downstream signalling - Deactivation and internalisation?

Answer 38

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

Question 39

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)

Answer 39

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

Question 40

What was used to get crystal structure of β2AR? (hint - chimera) How was inactive state stabilised?

Answer 40

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

Question 41

Through overlapping and comparing rhodopsin and β2AR structures, what differences were uncovered? (hint - lid, specificity)

Answer 41

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

Question 42

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)

Answer 42

Binding site has polar residues which interact with polar tail of ligand Aromatic/hydrophobic residues interact with ring structure in ligand

Question 43

As carazolol has very low Kd and slow off-rate, it has tight binding What structure makes this binding even tighter? (hint - sammidge)

Answer 43

Hydrophobic sandwich 2 hydrophobic layers enclose and 'sandwich' the hydrophobic parts of the ligand, making it tighter and less likely to release

Question 44

How are the receptor interactions different between full, partial agonists and antagonists?

Answer 44

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

Question 45

How is ionic lock broken in β2AR? (hint - rhodopsin)

Answer 45

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

Question 46

How can we crystallise β2AR in complex with a G protein?

Answer 46

T4L fusion and detergent micelles can be used to crystallise active receptor

Question 47

What are the differences between inactive and active state of β2AR? (hint - movement, hinge)

Answer 47

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

Question 48

How does Gα open and what does this allow?

Answer 48

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

Question 49

What is an analgesic? What receptor do many analgesics bind? What trait isn't ideal?

Answer 49

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

Question 50

How did they test many molecules to assess their binding to α2A-Adrenergic Receptor (α2AAR)? (hint - DOCK) What does the score depend on?

Answer 50

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

Question 51

What traits did new agonists found for (α2AAR) via docking tests show?

Answer 51

Low nM to low μM binding constants - Need even tighter binding All have 6C rings as well as linker groups between rings - Similar features

Question 52

What did docking derived agonist show that other drugs didn't?

Answer 52

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

Question 53

What was used to optimise initial docking hits? (hint - cryo, EC50)

Answer 53

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