Multidrug Efflux from Bacteria

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Multidrug Efflux from Bacteria
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- Most Antibiotics must get to the cytoplasm to
be effective
Periplasm
Cytoplasm
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Method Number 1 ATP-Binding Cassette
transporters ATPases of the third kind
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Introduction

• ABC-ATPases constitute one of the largest and
  most highly conserved protein superfamilies
• Several representatives are present in all
  organisms
• Alias MDR (multi-drug resistance) and P-gp
  (P-glycoprotein
• P-gp is clinically relevant in resistance to
  chemotheraputic agents
• Similarly, ABC-transporters provide multi-drug
  resistance in bacteria
• Indeed, nearly 2 of all bacterial proteins
  recorded to date are members of this protein
  superfamily.

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Introduction cont.

• ABC-dependent transport can be inwards or
  outwards.
• In some, the ABC-ATPase can even fuel the gating
  of an ion channel (e.g. CFTR).
• Also, it may regulate a pathway at the
  protein-lipid interface.
• The incredibly diverse substrates transported via
  the ABC class is uncharacteristic among ATPases.
• More peculiar is that fact that even within a
  single transporter the transported substrates can
  differ.

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• Basic structural unit of an ABC-transporter
• Six transmembrane domains
• One ATP-binding domain
• w/ characteristic Walker A B binding motifs
• -This appears to be ½ of the functional unit
  (i.e. a functional dimer)

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A D Characteristic of Prokaryotic Importers E
F Characteristic of Prokaryotic Exporters I
L Characteristic of Eukaryotic Exporters
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X
Normal transporters substrate specific
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How can P-gp can transport all of these molecules?
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The transport site is set up to recognize general
characteristics at various distances on a
molecule rather than specific substituents (e.g.
hydrophobicity 10 A away from a positive charge)
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Models of Transport
1. Aqueous Pore
2. Hydrophobic Vacuum
3. Flippase
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This degree of structural resolution doesnt
really help answer the mechanistic questions
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A picture is worth a thousand words!
Structure of MsbA from E. coli A homolog of the
Multidrug Resistance ATP Binding Cassette (ABC)
Transporters Geoffrey Chang and Christopher B.
Roth Science 293 1793-1800, 2001
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Methods

• Why did they choose MsbA?
• they didnt! It chose them.
• They cloned, expressed, purified 20 ABC
  transporters
• Membrane proteins are difficult to crystallize
  due to the separate solvent requirements of the
  lipid and aqueous phases
• Thus, they had to set-up numerous screens for
  each protein to hopefully obtain a suitable
  condition
• Specifically, they did 96,000 different screens!

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• Final Conditions were (grown in 50L fermentor)
• Extraction from E. coli with 1
  dodecyl-a-D-maltoside (DDM)
• Purified via Ni-NTA column in 20mM Tris (pH
  7.5), 20mM NaCl, and 0.05 DDM.
• Concentrated to 10-15 mg/ml pure protein
• Used Hanging drop method with 1 to 31 ratios of
  protein to precipitant soln.
• Checked screens daily and found initial formation
  at 3 weeks and full grown after 2 months!

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• They did not achieve atomic Resolution (
• Only 6.2 A (can only resolve the a.a. backbone)
• Used OsCl3 (heavy metal) to increase diffraction
  and achieved 4.5A resolution in one crystal
• They summed the data from five diffractions to
  come up with structure
• Also, they were able to use some isomorphous
  replacement with coordinates from the
  crystallized ATP-binding domain from HisP

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Bombard the protein crystal mounted in a
capillary tube with Xrays and pick up the
reflection on a screen behind the crystal
Measure the intensity of the diffraction spots
and their relationship to each other to
determine the atom distribution of the sample.
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• - All the TMDs are tilted 30-40o
• which forms the coned shaped
• structure with 2 huge openings
• Opening is 25A wide and leads
• to a coned-shaped chamber in the
• interior of the molecule.
• The base at the cytoplasmic
• side is 45A wide.
• -The dimer is held together solely
• by contacts between helices within
• the outer membrane leaflet

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Simply a cartoon of the previous page clearly
identifying the site of the lipid bilayer and
showing a Lipid A molecule. You can see that the
molecule can access from the inner leaflet, but
the outer leaflet is blocked. Can we rule out a
model?
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The structure shows that TMD2 and TMD5 of the
separate subunits are at the entrance to
the transport site. Also, they show 3 distinct
intracellular domains which are highly conserved
among ABC transporters and are undoubtedly the
communication link between substrate
transport and ATP hydrolysis. However,
deciphering this communication will take higher
resolution in several different nucleotide bound
states of the molecule.
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• This space-filling model shows the concavity of
  the internal chamber
• All 12 helices appear to play a role in chamber
  formation.
• interestingly the chamber is lined with positive
  charges, which may
• play a role in transport.

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We still cannot rule out the release of substrate
to the extracellular Space rather than the upper
membrane leaflet (generally speaking)
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Method Number 2 Resistance-Nodulation-cell
Division RND family of efflux pumps
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RND pumps form complexes with 1) Periplasmic
proteins of the Membrane Fusion protein
family (e.g. AcrA). 2) Outer membrane Channels
(e.g. TolC) Thus, drugs are effectively
removed by being pumped directly into
the extracellular medium. Differ from ABC
transporters in that they energy comes via
proton-motive force, not ATP.
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• AcrA, AcrB, and TolC tripartite Efflux Complex
• Pumps out tetracycline, cholramphenicol,
  b-lactams, novobiocin, fusidic acid, nalidixic
  acid, and fluoroquinolones.
• -Also, removes SDS, Triton X-100, and bile salts
  and other amphiphiles.
• - Recent high-resolution structures have provided
  a wealth of new information for the function of
  these efflux mechanisms as well.

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One proposed mechanism. 1) Ligand binds to AcrB
2) Induced conformational change in AcrA 3)
pulls OM and PM together 4) TolC joins with
AcrB. 5) Ligand transferred to TolC and released
to extracellular space.
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• X-ray Structure of AcrB.
• - Functional Homotrimer
• - Each monomer has 12 TMDs
• - 1 huge periplasmic domain.
• The 3 periplasmic domains form a funnel-like
  structure.
• There is a large central cavity between the 3
  protomers (35A diameter)
• Just out side the external surface of the PM
  there is an opening between the subunits
  (called the vestibule) which leads to the central
  cavity

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Space filling model from the outside. - The box
shows the vestibule between two subunits.
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• Molecular Modeling
• - Homology models of AcrD and MexB were
  constructed based upon the crystal structure for
  AcrB
• Appears that the vestibule in AcrD is more
  acidic than AcrB, which may explain why AcrD can
  transport the positively charged aminoglycosides,
  whereas AcrB cannot.
• - Likewise in MexB a couple of acidic residues in
  AcrB have been replaced with basic residues,
  which may explain the slower transport rate of
  cationic antibiotics.

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• Still only half way there
• AcrB gets molecules out of the PM, but they still
  need to be exported out of the cell completely.
• It appears that ligands are scavanged from the
  periplasm or the periplasmic leaflet of the PM
• Still a Theory
• - But the very narrow top of AcrB is thought to
  fit into a channel formed by TolC. (i.e. section
  marked A)

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• Crystal Structure of TolC
• (Koronakis et al., Nature 405, 914,00)
• Also, a homotrimeric complex.
• The channel through the OM is produced via a 12
  panel anti-parallel b-barrel.
• 4 sheets are contributed by each monomer.
• - Huge extended a-helical stretch into the
  periplasm, which forms protected tube-like
  structure.
• - This downward extension of TolC and the upward
  extension of AcrB combined can easily span the
  periplasmic space.

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• b sheet structure. 
• only backbone atoms are shown, excluding
  hydrogens. 
• The two strands are anti-parallel.
•  
• - Beta barrel is a closed b sheet.  

The Anti-parallel alignment puts a hydrophobic
surface facing the lipid bilayer and a
hydrophilic central cavity.
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How does water cross lipid bilayers?

• Aquaporins
• Water channels that mediate the single file
  movement of H2O down its concentration gradient.
• Similar in structure to the cation channel
  formed by Gramacidin which mediates H movements
  as H3O.
• Why then dont aquaporins also move H?

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