ER - Antibacterials & Drug Efflux III

Question 1

What are the types of efflux pumps in bacteria? (6)

Answer 1

1. What are the types of efflux pumps in bacteria? (6ATP-binding cassette (ABC)
2. Multidrug and toxin extrusion (MATE)
3. Major facilitator superfamily (MFS)
4. Proteobacterial antimicrobial compound efflux (PACE)
5. Resistance-nodulation-cell division (RND)
6. Small multidrug resistance (SMR)

Question 2

What is the role of efflux pathways in bacteria under antibiotic stress?

Answer 2

Activated almost ubiquitously to combat antibiotic effects

Question 3

Which efflux pumps are found in mammalian cells? (3)

Answer 3

• Multidrug and toxin extrusion (MATE)
• Major facilitator superfamily (MFS)
• P-glycoprotein 1 (P-gp, also known as MDR1 or ABCB1)

Question 4

What are the three types of transport structures in mammalian efflux pumps?

Answer 4

• Uniporters
• Symporters
• Antiporters

Question 5

How do bacterial MDR efflux pumps utilize energy?

Answer 5

Use proton/sodium motive force for substrate efflux

• except for ABC family which uses ATP hydrolysis

Question 6

Why are efflux pumps considered promiscuous?

Answer 6

They enable bacterial cells to tolerate multiple antimicrobial agents, contributing to antibiotic resistance

Question 7

How do ABC transporters export drugs? (3)

Answer 7

• Transmembrane domains (TMDs) of ABC transporters comprise the substrate-binding pocket
• The nucleotide-binding domains (NBDs) bind and hydrolyse ATP to drive the transport cycle
• Conformational changes are linked to the dimerization and dissociation of the NBDs, which are mediated by ATP binding and hydrolysis

Alternating access model: alternate between inward- and outward-open states

Question 8

How do MFS transporters export drugs? (3)

Answer 8

• Conformational switch triggered by induced fit from ligand binding, with transport rate controlled by electrochemical proton gradients
• Substrate binding leads to a switch from outward-open (influx) or inward-open (efflux) to an occluded conformation of the dimer
• Dimer then opens to release substrate on the opposite side (inward or outward), followed by deprotonation of LacY

Rocker-switch model: involves substrate and H+ symport, exchange, and counterflow reaction

Question 9

How do MATE transporters export drugs? (3)

Answer 9

• Substrate and proton bind to different residues in the transporter
• Mechanism involves a sequential process: substrate binds after proton release at the inside membrane surface, and substrate is released before proton binding at the outside surface
• Some MATE multidrug transporters exhibit both direct and indirect mechanisms of competition

V-shaped central cavity: facilitates substrate and H+ or Na+ antiport, exchange, and counterflow reaction

Question 10

How do RND transporters export drugs? (3)

Answer 10

• Drugs enter the binding pocket in the periplasmic domain in the access (or loose (L)) state
• They progress into the drug-binding pocket in the binding (or tight (T)) state, then are extruded into a funnel-shaped ‘canyon’ in the docking domain in the extrusion (or open (O)) state
• State switching is associated with changes in protonation of side chains in the transmembrane domain (R1 & R2 = 5-helix repeats) and resulting TMH movements

Functional rotation mechanism: involves asymmetric monomers in RND integral membrane protein trimers

Question 11

Why are RND-efflux pumps considered the most promiscuous?

Answer 11

They have voluminous binding pockets that can accommodate a variety of substrates

Question 12

What is the prototypical example of RND-type pumps?

Answer 12

AcrB

• Known as the most promiscuous RND pump

Question 13

Describe the characteristics of the binding pockets in RND-efflux pumps.

Answer 13

Extensive distal binding pocket containing many hydrophobic residues, along with charged and polar ones

Question 14

What principle accounts for the polyspecificity of RND transporters?

Answer 14

Hydrophobic drugs do not need small, shape-complementary interaction surfaces for favorable interactions, as they are not strongly stabilized in bulk solvent and require less desolvation

Question 15

How do RND transporters demonstrate binding diversity?

Answer 15

They allow for many different binding positions and modes within their voluminous drug-recognition pockets

Question 16

How do drug channels facilitate drug entry? (3)

Answer 16

• They provide various pathways for entry; hydrophobic drugs reside in the outer leaflet of the inner membrane and enter the periplasmic pore via channels (1 and 3)
• Hydrophobic compounds tend to partition into the bilayer, making them good substrates
• Amphipathic substrates (channel 2) stabilize initial contact through side chain interactions, allowing a broad range of ligands to be accepted with minimal differences in binding energy

Question 17

How do RND transporters gain resistance through mutations? (3)

Answer 17

They can mutate to become more resistant; for example, the RND transporter class (e.g., AcrB) shows increased resistance in clinically, environmentally, and laboratory-evolved strains

• There is a notable rise in gain-of-function mutations in the binding pockets of homotrimeric RND multidrug efflux pumps
• Often, resistance arises from a single point mutation—just one residue change in a protein with over 1000 residues