CC3: How do cells use energy stored in gradients?

Question 1

How was the F1 component of ATP synthase discovered?

Answer 1

1. Mitochondria were broken down and sedimented in an ultracentrifuge.
   - Sediment could respire, but not synthesize ATP.
2. Supernatant added back to the sedimented fraction.
   - Restored oxidative phosphorylation.
3. Component of soluble fraction was isolated and named factor 1 of oxidative phosphorylation.

Question 2

Describe the structure of an F-type ATP synthase and where they are present.

Answer 2

• large enzyme complex with more than 20 chains and 8+ subunits
• 550-850kDa
• encoded by both nuclear and mitochondrial genes
• present in bacterial plasma membrane, mitochondrial inner membrane, and chloroplast thylakoid membranes

Question 3

What are the two ways by which the subunits in F-type ATPases can be classified?

Answer 3

1. Structure - based on Racker’s experiment (F1 and Fo)
2. Function - rotor and stator

Question 4

What is the binding change mechanism of ATP synthase?

Answer 4

The binding change mechanism of ATP synthase refers to the way in which the enzyme uses proton motive force to drive the synthesis of ATP from ADP and inorganic phosphate.

1. Binding of ADP and inorganic phosphate (Pi) to the catalytic sites of the F1 portion of the enzyme.
2. Conformational changes in the enzyme complex caused by the rotation of the c-ring in the F0 portion, which exposes the catalytic sites to the matrix or cytoplasmic side of the membrane.
3. Release of ATP from the catalytic sites and binding of ADP and Pi to begin the next round of ATP synthesis.

During the binding change mechanism, protons flow through the F0 portion of the enzyme from the intermembrane space or periplasmic space to the matrix or cytoplasmic side of the membrane. The energy of this proton motive force is used to drive the rotation of the c-ring, which in turn drives the conformational changes in the F1 portion of the enzyme necessary for ATP synthesis.

Question 5

How was it shown that ATP release is the step that requires energy?

Answer 5

Labelled oxygen was monitored for distribution following ATP synthesis. It was observed that the exchange of phosphate oxygens with water oxygens resulting from the reversible cleavage and resynthesis of ATP continued in deenergized submitochondrial particles. This suggested that the formation of the ATP occurred readily without energy input and that the equilibrium between bound ADP and Pi and bound ATP was around 1. The hypothesis was therefore that energy input served to drive the release of tightly bound ATP.

Question 6

What is the name of the motif commonly associated with proteins that bind ATP?

Answer 6

Walker A motif

Question 7

How was F1 rotation visualized?

Answer 7

A bacterial a3B3y complex was modified with polyHis tags on the b-chains and a single cys on the y chain (all other cys were removed). The polyHis tags enabled the complex to be anchored to a slide covered in nickel, and the single cys residue was used to bind a large actin filament to which a fluorescent label had been added. This showed the complex moving in discrete 120 degree rotations.

Visualization was then improved when gold beads were used, providing less drag compared to the actin filament.

This was repeated again but using magnetic tweezers to force the stator in the opposite direction and synthesize ATP.

Question 8

How was rotation of the entire ATP synthase visualized?

Answer 8

1. FRET in reconstituted liposomes, using GFP on the stalk and two fluorescent probes on c-ring and y subunits. ATP synthesis was initiated by a K+/valinomycin diffusion potential, allowing both c and y subunits to be monitored by FRET as the distance between the GFP and FRET partners oscillated.
2. Nanodiscs - ATP synthase was put on a nanodisc and linked to a nanorod. Under polarized light, the color of the rod is dependent on the angle of the rod. Can visualize the change in color during the rotation of synthase.

Question 9

How was it shown that Glu59 is a critical residue in c-ring coordination of ions?

Answer 9

A key to the mechanism of c ring torque generation came from early studies with the covalent inhibitor DCCD. DCCD at extremely low concentrations will specifically label c subunits, reacting with the carboxylate side chain of Glu59 (or a conserved aspartate) and inhibit the entire c-ring. Crystallography showed that when sodium was bound to the ion-binding site, DCCD interaction was blocked.

Question 10

How are ions translocated through the c-ring in Fo?

Answer 10

1. Ions translocate through the half-channel (via a-subunit conserved His) until they encounter open glutamate. This half-channel is an aqueous environment that makes it unfavorable to protonate the glutamate.
2. The ring then ‘wiggles’ in a ratchet-like mechanism, moving glutamate back and forth.
3. Whenever it goes backwards, it reaches the hydrophobic environment of the lipid bilayer where it’s no longer unfavorable for the glutamate to be protonated. i.e., glutamate forms the closed conformation.
4. The glutamate won’t move back into the aqueous environment once it’s been protonated. This drives rotation.
5. The c-ring rotates to reach the second half-channel, where the basic environment causes proton release.

The mechanism works in reverse for ATP hydrolysis.

Backwards rotation is prevented by the positive charge of the strictly conserved arginine.

Question 11

What is the role of magnesium ions in F1Fo ATP synthase?

Answer 11

Magnesium plays a critical role in stabilizing the ATP molecule as it is being synthesized. When ATP is formed in the F1 unit, it is immediately bound by magnesium ions, which help to stabilize the molecule and prevent it from hydrolyzing back into ADP and Pi.

Question 12

What is the role of the universally conserved arginine in the a-subunit of ATP synthase?

Answer 12

Helps prevent short-circuiting in the complex by creating a series of gates that limit the movement of protons through the complex and force them to undergo the required conformational changes for efficient ATP synthesis.

Question 13

What is the importance of pKa in the ATP synthase mechanism?

How is the pKa fine-tuned?

Answer 13

The pKa of the carboxyl group determines the pH at which it will be protonated, which in turn affects the efficiency of proton translocation across the mitochondrial inner membrane.

Conserved glutamate or aspartate residues help to lower the pKa of the carboxyl groups by providing an environment that stabilizes their negative charge.

Question 14

Why are the helices within the a subunit of ATP synthase horizontal?

Answer 14

In a horizontal helix, the two conserved residues (Arg and His) are able to interact with two adjacent c-subunits simultaneously at a fixed distance, irrespective of the ring diameter.

This would not be the case if the a-subunit helices were oriented vertically.

Question 15

What can be found in the center of ATP synthase c-rings in AFM structures, and why?

Answer 15

Lipids - prevents protons from flowing through.

Question 16

What determines the size of the c-rings in an organism?

Answer 16

Having different size c-rings allows for variability in the ion:ATP ratio.
- smaller the ring, the more efficient ATP synthesis
- larger the ring, the more energy can be harvested from ATP hydrolysis to build up an ion gradient.

Question 17

What dictates the direction of ATP synthase activity? i.e., ATP hydrolysis vs ATP synthesis.

Answer 17

Depends purely on the thermodynamic balance between the PMF and the Gibbs free energy change for ADP phosphorylation.

Question 18

How is ATP synthase regulated by:
- ADP
- IF1
- Chloroplast y-subunit
- Bacterial e-subunit
- a-Proteobacterial E-subunit
-

Answer 18

ADP: when PMF is low, ADP-Mg (without Pi) remains bound to one of the catalytic sites to form an inactive complex.

IF1: an inhibitory homodimer (can inhibit two ATP synthases at once) that binds between a-DP and B-DP subunits.

Chloroplast: y contains a disulfide bond that forms at night to prevent ATP hydrolysis when there’s little PMF.

Bacteria: e-subunit protrudes between the B and y subunits to inhibit ATP hydrolysis for PMF formation. When e-subunits folds down, ATP hydrolysis can occur.

a-Proteobacteria: inhibits ATP hydrolysis in a similar mechanism to IF1.

Question 19

How does IF1 regulate ATP synthase?

Answer 19

The N-terminus of IF1 is intrinsically disordered when unbound, but folds into an alpha helical structure upon binding ATP synthase.

Hydrolysis of one ATP partially folds the IDP region, and a second hydrolysis converts it into the fully closed state. This stops ATP hydrolysis from going any further.

In the presence of PMF, the bound IF1 is released and ATP synthesis can resume.

Question 20

Why do mitochondrial ATP synthases form dimers in vivo?

Answer 20

Studies showed that removal of ATP synthase dimers results in mitochondrial vesicles lacking curved regions. This has seen been shown in mitochondria, demonstrating that dimerization of mitochondrial ATP synthase helps to curve the membrane.

Question 21

What diseases are associated with ATP synthase?

Answer 21

1. NARP: mutations in mtDNA impact Fo function
2. MILS: neurological disorder with early death, caused by mtDNA mutations.
3. FBSN: neurological disorder
4. LHON: degeneration of retinal ganglion cells, causing vision loss

Question 22

How does Bedaquiline act against Mycobacteria?

Answer 22

Treatment against tuberculosis.

Binds at the proton binding site within the c-ring (similar to DCCD and oligomycin), and prevents rotation due to how bulky it is.

Specificity comes from the sequence differences that produces a different shape of hydrophobic residues e.g., no Phe in mycobacterium.

Question 23

Compare F-type, A-type (and A/V-type) and V-type ATPases.

Answer 23

F-type:
- 1 outer stalk
- ATP synthesis and hydrolysis in eubacteria and eukaryotes
- potential drug targets

A-type and A/V-type:
- 2 outer stalks
- ATP synthesis and hydrolysis in archaea (A) and eubacteria (A/V)

V-type:
- Up to 3 outer stalks
- ATP driven ion pump in eukaryotes
- Relation to diseases e.g., cancer (they can be upregulated to acidify the ECM and cause ECM degradation for tumor invasion and metastasis)

Question 24

Give an example of proton pumps in a yeast cell and a plant cell.

Answer 24

Yeast:
Pma1 pumps protons out of the cytosol and into the ECM at the expense of ATP.

Plant:
AHA (same as Pma1)

Question 25

How are eukaryotic V-type ATPases regulated?

Answer 25

One major regulatory mechanism is reversible dissociation of the V1 and V0 domains. This can be achieved through post-translational modifications of the subunits, such as phosphorylation and acetylation, which affect the stability of the interaction between the two domains.

Question 26

Describe the structure and mechanism of Pma1 in yeast.

Answer 26

A P-type ATPase (A, P, and N domains) that forms hexamers.

P-type ATPases go through a conformational cycle described by the E1/E2 Post-Albers scheme, with E1 denoting states with a high affinity to the proton.
The proton-acceptor/donor in Pma1 is the conserved aspartate residue in M6. This residue has been shown to be essential for proton transport and E1  E2 transitions in Pma1. There’s also an equally highly conserved asparagine in M2 that is at a distance suitable for capturing a proton within a hydrogen bond. Proton release to the extracellular side has been suggested to be driven by a conformational change that breaks this hydrogen bond and instead allows the formation of a salt bridge between the deprotonated aspartate and an adjacent arginine residue, whilst also opening up the exit pathway.

Question 27

Describe the function of the human gastric ATPase.

Answer 27

Counter-transports K+ ions with protons within the membrane of gastric parietal cells. It pumps protons into the stomach lumen to create a pH of 1 to digest food.

Question 28

What are the ‘other’ uses of the PMF (apart from ATP synthesis)?

Answer 28

Energy source for:
- Cation import
- H+ coupled secondary transport
- SEC system
- Bacterial efflux pumps

Acidification of compartments:
- Protein trafficking + cargo sorting
- Lysosomal enzymes
- Gastric acid

Question 29

How do animal cells energize their plasma membrane?

Answer 29

They use sodium ions, rather than protons.

Question 30

Describe two methods that can be used to visualize bacterial movement.

Answer 30

1. Swarm plate:
   Bacteria are spotted on in small dots, allowing for the visualization of bacteria moving away from the spot.
2. Microscope + capillary tube:
   Capillary tube filled with chemical that attracts bacteria, allowing for visualization of bacteria moving to and congregating around the capillary - movement with purpose.

Question 31

Describe the structure and distribution of flagella in E. coli.

Answer 31

Distribution:
- Peritrichous (randomly distributed)
- 5-8 per bacterium

Structure:
- 3 parts: basal body, hook and filament
- Basal body is the motor
- Hook connects the body to the filament
- Filament is what rotates to propel the cell

Question 32

Describe the structure of the bacterial flagellar filament.

What are L- and R-types?

Answer 32

• Composed of flagellin subunits
• 11 subunits per turn
• Hollow, with a central channel that runs the entire length of the filament

L- and R-type filaments refer to the left-handed and right-handed helices the filaments can be made from. The L-type filaments are composed of flagellin, whereas R-type filaments are composed of two proteins: FlaA and FlaB.

Question 33

Describe the structure of the bacterial flagella hook and basal body. State the subunits they’re made from and the functions.

Answer 33

Hook:
- Universal joint
- FlgE

Basal body:
- Stator and a rotor
- Rotor is composed of a central rod (FliE) which extends from the center of the rotor, surrounded by rings of FliG, FliM and FliN
- Stator is stationary and surrounds the rotor, functioning as a channel for ions to provide energy for the motor. Composed of MotA and MotB (E. coli).
- Switch compex (FliG, FliM, FliN, CheY) controls the direction of flagellar rotation.

Question 34

How is the bacterial flagellar motor powered?

Answer 34

Ion motive force.

The flow of ions through the motor causes a conformational change in the stator protein (e.g., MotA) which then rotates the rotor protein (e.g., FliG). This rotation is transmitted to the flagellar filament, causing it to spin and propel the bacterium.

Question 35

What is the stoichiometry of the stator subunits, and why is this believed to be important?

Answer 35

MotA (5) : MotB (2)

Thought to be important to create half-type channels for ion translocation. Without this ratio, the mechanism for ion translocation wouldn’t work.

Question 36

What is charge reversal in the context of the bacterial flagellar motor?

Answer 36

Refers to the process of FliG flipping in the y-axis such that it interacts differently with MotA and hence direction of flagellar rotation is switched.

Question 37

Why is MotB thought to interact with the peptidoglycan layer?

Answer 37

It gives it something to ‘push against’ and causes the plug domain to be removed from the channel, showing the MotAB stator to be active. When not bound to the PG, the plug domain blocks the channel.

Question 38

Describe the resurrection experiment, used to determine the number of stators in bacterial flagella.

Answer 38

A bacterial strain with mutations in MotA or MotB is transformed using IPTG to induce expression of WT stator complexes.

The output is then measured, looking for steps up in the speed of rotation. The assumption is that every step up is the addition of a stator subunit.

Question 39

What experiments were used to show that stators are exchanged?

Answer 39

TIRF, photobleaching and fluorescent recovery showed this exchange occurs.

Changing the salt concentration in sodium-driven motors then showed that increasing the concentration of salt meant more stators become engaged with the motor.

Engineered cells with light-driven proton pumps showed that the stators are added in discrete steps depending on the amount of IMF available.

Question 40

How does viscosity impact stator number?

Answer 40

By measuring the rotation of a bead in different media, it was shown that increasing the viscosity resulted in an increase in the number of stators engaged with the motor.

We don’t know how this is sensed.

Question 41

Describe how conformational spread in FliG is activated and leads to switching of direction in the bacterial flagellar motor.

Answer 41

1. Binding of a signal molecule to a MCP receptor that is normally methylated by CheR causes activation of CheA.
2. CheA activates CheB which demethylates MCP.
3. CheA phosphorylates CheY.
4. CheY binds the FliM and induces a conformational change in FliG to switch the relative positions of the two conserved charged residues, changing direction of flagellar rotation.
5. FliG must all be in one orientation or the other, so conformational spreading occurs across all FliG proteins.

Question 42

Describe how the motor is made in E. coli.

Answer 42

1. FlhDC are transcribed to regulate transcription of a sigma factor (sigma 28).
2. Sigma 28 turns on genes for the motor. FlgM is an anti-sigma factor to prevent this.
3. Once the basal body is built, FlgM is secreted to allow full activation of sigma 28 and turning on of the filament genes.

This means that if the basal body isn’t complete/functional, the rest of the flagellar isn’t built.

Question 43

How is hook length determined in bacterial flagella?

Answer 43

FliK acts as a molecular ruler.

During hook assembly, FliK blocks the channel and determines whether its N and C termini are able to bind the hook cap and basal body, respectively, at the same time. This measuring isn’t continuous due to FliK blocking the channel.

Once this occurs, there’s a switch to export FlgM (anti-sigma factor) so that sigma28 can start transcribing filament genes.

Question 44

Describe the export system used to make the bacterial flagella filament.

Answer 44

This system contains a chaperone protein (FliH) and an export protein (FliI) that are homologues of ATP synthase subunits.

ATP is needed for the docking of cargo, rather than the export itself.

Question 45

How can bacteria adapt to sense and ‘remember’ their environment via their flagella?

Answer 45

MCPs (chemoreceptors) can be methylated to reduce their affinity for the ligand they recognize. The methylation state of the receptor acts as a short term memory for the bacteria to determine whether the environment is better or worse.

Question 46

What is bacterial twitching?

Answer 46

Bacterial twitching is a form of bacterial motility that involves the extension and retraction of type IV pili (TFP), which are thin, flexible appendages present on the cell surface of many bacterial species. The TFPs extend out from the cell body and attach to a substrate, and then contract to pull the cell forward. This process can be repeated multiple times to generate movement in a directional manner. Bacterial twitching is thought to be particularly important for bacterial movement on solid surfaces, such as soil, rocks, and the surfaces of animal tissues.

Question 47

List processes that depend on energy transduction through the bacterial cell envelope.

Answer 47

1. Gliding motility
2. Bacterial flagella rotation
3. Extracellular protein secretion (Type VI)
4. LPS secretion
5. Nutrient import

Question 48

What is the PEZ model of LPS deposition?

Answer 48

As LPS molecules are being pushed into LptD, it fills the channel, pushing those at the top of the channel onto the surface of the membrane (like a PEZ candy dispenser).

Question 49

How is LPS secreted in Gram-negative bacteria?

Answer 49

Lipid A is synthesized in the cytoplasmic membrane. Flippase flips the molecule into the periplasm. An ABC transporter extracts the lipid from the membrane and the LPS is moved up to the OM via the PEZ model.

LptDE inserts LPS into the membrane via a lateral gate.

Question 50

Which processes can be driven by PMF in the outer membrane of Gram-negative bacteria?

Answer 50

TonB-ExbB-ExbD - molecule uptake
TolB-Pal - divisome assembly
Lol pathway - lipoprotein insertion
BFM - movement

Question 51

How is the PMF coupled to the TonB box plug removal in the Ton system?

Answer 51

ExbB-ExbD complex is a 5:2 assembly, so is predicted to act in the same way as MotAB (even has the conserved aspartate).

Pentamer rotates around the central dimer, using the PMF to generate a force that can energize TonB to open TBDTs.

Question 52

Describe the Ton system in Gram-negative bacteria.

Answer 52

The Ton system (composed of TonB, ExbB and ExbD) interacts with Ton-dependent receptors e.g., those that bind siderophores.
- ExbB and ExbD sit in the IM (PMF)
-TonB sits in the PG
- TBDT sits in the OM

The elongated TonB subunit physically interacts with nutrient bound TonB-Dependent Transporters (TBDTs) at the outer membrane. ExbB and ExbD harness the proton motive force (pmf), and transfer it to TonB which then opens a gate in the TBDT, allowing the nutrient to enter the periplasmic space.

There’s a TonB box that forms a perfect plug within the TBDT channel. This is somehow removed to open the channel.

Question 53

Describe the Tol-Pal system in Gram-negative bacteria.

Answer 53

Recruited to cell division sites where it uses the PMF to stabilize the outer membrane.

The TolQ-TolR (5:2) stator and TolA are free to diffuse within the inner membrane. Pal is bound to the mDAP moiety of PG unless in complex with TolB, which blocks PG binding and increases Pal diffusion in the OM. TolA associates with the stator, and PMF results in TolA to extend through the holes in the PG layer. At the OM, TolA binds TolB which is in complex with Pal.

Loss of protonation (PMF) causes the whole assembly to relax back to its starting position, providing the driving force to bring TolB down through the PG layer. TolB dissociates from TolA and diffuses to rebind Pal.

In a dividing cell, the stator-TolA complex is recruited to the divisome to confine TolB capturing activity. TolB-Pal complexes dissociate, so that Pal is located at the divisome and kept free of TolB.

Question 54

What is B-strand augmentation? Which bacterial membrane systems use it?

Answer 54

Beta-strand augmentation is a process by which a protein can add a beta-strand to an existing beta-sheet.

Ton: TonB partially unfolds TBDT beta-barrel to expose a B-strand (TonB-box plug) which allows PMF to be coupled to transport.

Tol: beta-barrel of TolB exposes a B-strand to interact with the beta-strand of TolA.