Drug Binding

Question 1

The binding pocket

Answer 1

• polyspecific
• many side chains are exposed into the pocket
• a subset of these side chains are involved in binding a specific substrate
• plasticity is important as the pocket is so large
• weak molecular interactions between amphiphilic drugs and MDTs
• weak interactions are similar to ligand interactions in iono- and metabotropic receptors but are organised within these large binding chambers

Question 2

Weak molecular interactions within the binding pocket

Answer 2

hydrogen bonds
salt bridges
carbon-pi interactions
- donors and acceptors in the right position

hydrophobic interactions
van der Waals force
- complementary surfaces

Question 3

Energy coupling

Answer 3

• helix rotations in P-gp dictate binding affinity
• switch from in to outward-facing
• with rotation of transmembrane helices
• rotation causes side chains to move away from the cavity
• disrupts favourable interactions
• reduced binding affinity in outward-facing conformation allows ligand dissociation

Question 4

Energy coupling - ABCs

Answer 4

• conformational changes in ABCs are controlled by ATP binding and hydrolysis
• homodimers bind and hydrolyse 2ATP
• heterodimers bind 2ATP but only hydrolyse 1ATP

Question 5

Energy coupling - AcrB trimer

Answer 5

• antibiotics first bind proximal pocket which collapses and forces antibiotic into the distal pocket
• access –> binding –> extrusion

Question 6

ABC structure

Answer 6

• conserved architecture
• 2NBDs and 2TMDs
• 2NBDs are linked by coupling helices and forming 2 nucleotide binding sites at the dimer interface

Question 7

MDT Bmr

Answer 7

• MFS family
• antiport with protons
• Bmr co-expressed with BmrR

BmrR
- transcriptional activator
- sensor for antibiotics
- switches on Bmr when bound to antibiotics
- to mediate efflux through Bmr

• drug selectivity of receptor and transporter overlap
• regulator crystallised first
• BmrR binds operational sequence
• role of acidic residues for BmrR = flap opens to expose groove and allow cation to bind and activate

Question 8

Physiology - redundancy

Answer 8

more than 5RND efflux pumps in E.coli = redundancy and overlapping specificity - WHY?

Question 9

Physiology - role in protection

Answer 9

• in vivo bile acid resistance in Enterobacteriaceae
• RND systems present in gram -ve bacteria
• greater drug susceptibility between E.coli acrAB mutants and their isogenic wt strains (reduced MIC)

Question 10

Physiology - role in quorum sensing

Answer 10

• MDTs can mediate efflux of signalling molecules
• more MDTs = more more efflux of signalling molecules = threshold reached earlier = switch on transcription of stationary phase genes

Question 11

Physiology - role in lipid transport

Answer 11

• in gram -ve bactera
• ABC transporter MsbA can transport drugs and also lipid A and PE
• other studies show that MDTs can transport a variety of lipids and lipid-soluble compounds