Receptors and fusion proteins

Question 1

What are some features of ionotropic receptors?

Answer 1

Ligand gated
<50ms speed
All or nothing
Multiple interchangable subunits
Neurotransmitter sensor
Changes membrane potential

Question 2

What are some features of metatropic receptors?

Answer 2

GPCR
100ms to minutes
can amplify/dampen signals
Generally monomeric

Question 3

Describe glycine receptors

Answer 3

Fast inhibitory neourotransmission at synapses
Motor control and pair processing in CNS
5 subunits
Inhibited with picrotoxin and imaged without ligands
Glycan density at Asp62 affects how the protein affects ER

Question 4

Describe nAchR structure

Answer 4

Cys-loop ion channel
Pentameric
Lots of TM helices, hydrophobic outside, hydrophilic inside
Ach binds two sites at interfaces of each α subunit
C-loop closes around the ligand

Question 5

How are Cl- channels gated?

Answer 5

Glu receptors which move Cl- out of the cell to inhibit polarisation of the membrane

Question 6

What helps GluCl crystallise?

Answer 6

Fab antibody fragements bind the channel at 5 sites which are (+)

Question 7

How does the GluCl channel open?

Answer 7

β1-β2 loops twist and tilt M2 and M3 away from the central axis, opening the core

Question 8

What is transducin?

Answer 8

GPCR which has rhodopsin as its G-protein

Question 9

What is rhodopsin made up of?

Answer 9

Opsin and retinal (chromaphore)

Question 10

Describe transducin activation

Answer 10

1. One photon activates retinal which gets transducin in an activated state
2. Gα-GDP is exchanged for Gα-GTP
3. GTP is hydrolysed to switch off the signal
4. Membrane is hyperpolarised by Na+ import and Ca2+ export which is interpreted by the brain as light

Question 11

What structural features do transducin have?

Answer 11

Amphiphilic domain near C-terminus
Conserved sequences on TM3 and TM6/7 form an ionic lock.
TM6 swings open when light is absorbed, allowing Gαt
At ground state a lid prevents access to a rhodopsin binding site, gated by ionic lock between TM6 and 7

Question 12

What happens when retinal absorbs light?

Answer 12

Energy pushes against the protein, breaking the ionic lock and allowing rhodopsin to bind to transducin
11-cis to all-trans retinal
Gt preferrentially binds Meta II to induce transducin activity

Question 13

How are transducin intermediates trapped for study?

Answer 13

1. Retinal removed, add Gαt fragment to have Meta II conformation
2. Mutants with consituitive activity, e.g disrupting the counterion to keep it in an all-trans conformation

Question 14

How are α2A adrenergic receptors targetted for CNS pain relieving effects.

Answer 14

Helical movements trap Gα in the active state
Using a virtual molecule library (ZINC15) which scores molecules based on van der waals and electrostatic interactions

Question 15

How are β2AR locked in an open conformation?

Answer 15

Carazolol is an inhibitorwhich sits in a binding site with low Kd and slow off rate

Question 16

Describe β2AR activation

Answer 16

1. Adrenaline binds to the β2AR. TM5 and 6 open up which allows G protein to bind
2. Gs activates adenylate cyclase, converting ATP to cAMP
3. cAMP activates L-type Ca2+ channels on the ER to release Ca2+
4. PKA is activated, eliciting downstream effects

Question 17

What inactivates β2AR?

Answer 17

BARK (type of GRK)
Arrestin
PKC

Question 18

Describe the nuclear pore

Answer 18

Found at inner and outer junctions of nuclear envelope
30 different nucleoporins
Aids in DNA histone synthesis in cytosol
Expands when a peptide sequence is recognised
Transport guided by exportins, importins and Ran-GTP

Question 18

What are fusion proteins used in?

Answer 18

Fusion of organelles
Plasma membrane repair
Vacuole fusion
Viral entry
Exocytosis

Question 18

What forces prevent membranes spontaneously fusing?

Answer 18

Membrane proteins repelling each other
Polar lipid heads repelling each other and attracting water (repulsive hydration force)
Membrane movement creating an entropic barrier

Question 18

Outline how two haemaglutanin molecules form a membrane pore in the host cell membrane

Answer 18

1. Haemaglutanin overcomes energetic barriers and recognises cell receptors. The receptor is taken up by the endosome via endocytosis
2. pH5 in the endosome causes haemaglutanin heads (HA1) to become loosely tethered. α helix forms and kicks the N-terminus to the host cell membrane
3. HA0 is proteolytically cleaved to HA1 and HA2 for membrane fusion.
4. Pore forms

Question 19

Why is haemaglutanin proteolytic cleavage irreversible?

Answer 19

Free energy is low after cleavage

Question 20

How is the SARS COV-2 spike protein embedded into host cell?

Answer 20

TMPRSS enzyme from host cell cuts spike protein which reveals hydrophobic amino acids that allow it to embed into the host cell membrane

Question 21

Describe the SNARE complex

Answer 21

Coiled coil made up of 4 α helix strands
Main proteins are SNAP and Syntaxin
Bonded by heptad repeats which form hydrophobic interactions
Fuse vesicles to membranes
Syntaxin drives membranes closer together
Zero layer has 3Q:1R ratio conserved across species
Dynamin pinches off the vesicles

Question 22

What recycles SNAREs?

Answer 22

NSF which binds to SNAP receptors to inhibit membrane fusion.

Question 23

What is the HSV gB fusogen complex made up of?

Answer 23

gD, gB, gH/gL

Question 24

How can HSV (Herpes simple virus) spread cell to cell

Answer 24

Glycoproteins gE/gI accumulate in the host trans-golgi network so they can attach to another cell’s receptors and cause virus spreading

Question 25

Describe how gB joins HSV and host cells

Answer 25

1. gD binds to its receptors on the host cell and undergoes a conformational change
2. The signal is relayed to the gH/gL. gH tail wedges in the gB CTD clamp
3. gB is released and its fusion loop binds to the host’s surface (easier as gB has lots of hydrophobic amino acids)
4. Post fusion gB complex forms a hemifusion stalk and completes membrane fusion to form the nuclear pore