Alistair Flashcards
What are the names of the two efflux pumps?
AcrAB-TolC
MacAB-TolC
How are the two pumps powered?
AcrAB-TolC = proton motive force MacAB-TolC = atp hydrolysis
What is the structure of TolC?
Trimeric
50kDa.
Porin.
β-barrel of 3x 4 antiparallel strands forms a pore in the outer membrane.
The top (outer membrane) remains open all the time but the bottom is closed (on the periplasmic side).
The tips of the helices come together to close at the bottom to differentiate between periplasm and outside world.
Describe open and closed states of TolC
In absence of the rest of the machinery, TolC is closed. It opens with conformational changes. It only happens very rarely, only when the entire thing is assembled. Makes sense – to stop leakage/invasion.
Hydrogen bonds seal the periplasmic end.
It opens when in tripartite system.
Describe periplasmic adaptors
- Long alpha helical hairpin domain interacts with TolC.
- Four domains like beads on a string with flexibility between them
- allows adaptors to wrap around TolC on OM and inner membrane pump to form a seal between the two.
- Anchored to the inner membrane with a cysteine residue with a fatty acyl group in the membrane (AcrA) or with a TM helix (MacA)
Structure of AcrB inner membrane pump
Trimer (three molecules of the AcrB monomer).
12 Transmembrane helices per monomer (36 total).
No pore so substrates are periplasmic.
Uses proton gradient.
Energy is used in Funnel domains.
What is the stoichiometry in AcrAB-TolC and how was it found?
3:6:3
Daurey et al (2016) Nature worked out the TEM (CryoEM) structure at low resolution of the entire complex.
Used nanodisc technology (a way of purifying membrane proteins in natural state) to keep pump in a sort of membrane physiological environment
How does AcrAB-TolC work?
Functional Rotation - lock-step mechanism by Murakami (2006). Trimer has three states: L = Loose, T = Tight, O = Open. Substrate binds, converts from L to T. Proton binds, converts from T to O.
What does knocking out MacB make a bacteria susceptible to?
macrolide resistance
Why do we look into MacAB-TolC?
Overexpressing it makes bacteria becomes tolerant to 32x more erythromycin.
Also provides resistance to Kanamycin, Colistin and Bacitracin.
Colistin is a last resort antibiotic. Only used for multidrug resistance. Bad side effects.
So resistance to this is scary.
Describe MacB structure
Crow (2018) solved structure with ATP.
Found a dimer where each monomer has multiple domains. 4 TM helices go through IM. Large domains in periplasmic space and cytoplasm. Nucleotide binding domains in cytoplasm.
How is MacB different from other ABC transporters?
It doesn’t move substrates across the IM but still provides resistance to antibiotics. We know this because little to no TM cavity compared to large cavities in others. Only enough space for a few water molecules.
Which toxin does MacAB-TolC secrete?
Enterotoxin STII even tho its produced in the cytoplasm! Knocking it out prevents secretion.
How does MacAB-TolC secrete toxins?
Enterotoxin STII has signal peptide targeting it to IM. It secretes via Sec, peptide is cleaved by SP and DSBA system adds disulphide bonds which lets it be recognised by MacAB-TolC. Knocking out cysteines prevents secretion, as is blocking ATP hydrolysis
Where might MacB be binding erythromycin antibiotic?
Mutating the periplasmic domain reduces antibiotic resistance so it must bind there.
