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Abc seminar
1. ABC OR
ATP BINDING CASSETTE
TRANSPORTER
BY
JASKAMAL GILL
M-PHARM (PHARMACOLOGY)
2nd semester
2. Multiple drug resistance or Multidrug
resistance is a condition enabling a disease-
causing organism to resist drugs or chemicals of a
wide variety of structure and function targeted at
eradicating the organism.
Organisms that display multidrug resistance can
be pathologic cells, including bacterial and
neoplastic (tumor) cells.
Multiple drug resistance
3. MECHANISMS INVOLVED IN MDR
Enzymatic degradation
Mutation at binding site
Down regulation of outer membrane proteins
Efflux pumps
Transduction of genes
4. Enzymatic degradation
Mechanisms of b-lactamase
N
O
N
O
OH
S CH3
CH3
O
R
H
b-lactamase
CH2
OH
b-lactamase
CH2
OH
N
O
N
O
OH
S CH3
CH3
O
R
H
b-lactamase
CH2
O
H H2O
N
O
N
O
OH
S CH3
CH3
O
R
H
H
OH
b-lactamase
CH2
OH
+
Hydrolysis of Oxyimino group
Penicillin drug
Inactivated drug
5. Mutation at binding site
In this binding of p53 to MDR1 is blocked at site(i.e. p53
DNA-binding site) and this mutation results in enhancement
of metastasis and mediate MDR
6. Down regulation of outer membrane
proteins
The outer membrane permeability is regulated by porin
proteins.
Alteration in Outer membrane permeability
particularly due to the decreased expression of porin
proteins results in decreased influx of various drugs.
Additional resistance is also afforded by over-expressed
efflux pumps that extrude a wide variety of unrelated
drugs which ultimately results in a multidrug resistance
(MDR).
7. EFFLUX PUMP
MDR is associated with increased expression of ABC drug
transporter P-glycoprotein (P-gp).
Pgp, the product of MDR1 gene is a membrane protein
consist of two duplicated halves each consist of
hydrophobic membrane spanning segments.
Two close genes i.e. MDR1 and MDR3 are located at the
long arm of chromosome 7 that encodes Pgp(30) .
MDR3 is not involved in drug resistance.
9. Serve as channels to transport molecules across cell membranes.
Facilitate the import of nutrients into cells or export toxic products into the
surrounding medium, which are essential for cellular homeostasis, cell growth, cell
divisions, and bacterial immunity.
Hydrolyze ATP and use it to move molecules against the concentration gradient or
transport substrates across lipid membranes.
Multi-domain structures that are comprised of two α-helical transmembrane domains
(TMDs) and two nucleotide-binding domains (NBDs).
Understanding ABC transporters’ structure and mechanism may help design agents to
control their function, which might be used to treat cancer and drug-resistant bacteria.
ATP-binding cassette transporter
10. ATP-binding cassette transporter
• ABC transporters transporter molecules such as
-ions, sugars, amino acids, vitamins, peptides, polysaccharides, hormones,
lipids.
• ABC transporters are involved in diverse cellular processes such as
- maintenance of osmotic homeostasis,
- nutrient uptake,
- antigen processing,
- cell division,
- bacterial immunity,
- pathogenesis
11. The 48 human ATP-binding cassette (ABC) genes have been
subdivided into seven subfamilies from ABC-A to ABC-G based
on their relative sequence similarities. Subfamily C contains
thirteen members and nine of these drug transporters are
often referred to as the multidrug resistance proteins (MRPs) .
Of the nine MRP proteins, four, MRP4, -5, -8, -9 (ABCC4, -5, -
11 and -12), have a typical ABC structure with four domains,
comprising two membrane spanning domains (MSD1 and
MSD2), each followed by a nucleotide-binding domain (NBD1
and NBD2) and these are referred to as the ‘short’ MRPs.
Introduction
12. The so-called ‘long’ MRPs, MRP1, -2, -3, -6, -7 (ABCC1, -2, -3, -6
and -10), have an additional fifth domain, MSD0, at their N-
terminus.
MSD1 and MSD2 form the translocation pathway through
which substrates cross the membrane, whereas the two NBD
proteins associate in a head-to-tail orientation to form a
‘sandwich dimer’ that comprises two composite nucleotide
binding sites.
In prokaryotes, importers mediate the uptake of nutrients into
the cell. The substrates that can be transported include ions,
amino acids, peptides, sugars, and other molecules that are
mostly hydrophilic.
Eukaryotes do not possess any importers.
Exporters or effluxers, which are both present in prokaryotes and
eukaryotes, function as pumps that extrude toxins and drugs
out of the cell.
13. FAMILY NAME NUMBER OF FAMILY MEMBERS LINKED HUMAN DISEASE
ABC A 12 Tangier disease (defect in
cholesterol transport)
ABC B 11 Bare lymphocyte syndrome type
I (defect in antigen-presenting)
ABC C 13 Dubin-Johnson syndrome (defect
in biliary bilirubin glucuronide
excretion)
ABC D 4 Adrenoleukodystrophy (a
possible defect in peroxisomal
transport or catabolism of very
long-chain fatty acids
ABC E 1
ABC F 3
ABC G 5 Sitosterolemia (defect in biliary
and intestinal excretion of plant
sterols
ABC SUPERFAMILY
18. Most transporters have transmembrane domains that
consist of a total of 12 α-helices with 6 α-helices per
monomer.
The TM domains are categorized into three distinct
sets of folds: type I ABC importer, type II ABC
importer and ABC exporter folds.
The type I ABC importer fold was originally observed
in the ModB TM subunit of the molybdate
transporter.
The type II ABC importer fold is observed in the
twenty TM helix-domain of BtuCD.
Transmembrane domain (TMD)
19. The ABC domain consists of two domains, the catalytic
core domain and a smaller, structurally diverse α-
helical sub domain that is unique to ABC transporters.
The larger domain typically consists of two β-sheets
and six α helices, where the catalytic Walker A motif
and Walker B motif is situated.
The helical domain consists of three or four helices and
the ABC signature motif.
The ABC domain also has a glutamine residue residing
in a flexible loop called Q loop. The Q loop is presumed
to be involved in the interaction of the NBD and TMD.
Nucleotide-binding domain (NBD)
21. BtuCD Structure and Mechanism
BtuCD is the vitamin B12 importer of
E.coli.
The crystal structure of BtuCD
provides the high resolution
visualization of an ABC transporter.
Bacterial ABC transporters use peri-
plasmic binding proteins (PBPs) to
capture substrate and present it to the
membrane translocator units.
BtuF is an example of PBPs.
BtuF deliver vitamin B12 to BtuCD
22. BtuCD Structure and Mechanism
In BtuCD, two copies of BtuC subunit and two BtuD subunits assemble to form the
functional heterotetramer, the BtuC2D2.
BtuCD structure revealed three features for ABC transporter function:
• the translocation pathway
• the arrangement of ABC domains
23. Translocation Pathway of BtuCD
Present at interface of the two BtuC
subunits
large cavity that opens to the peri-
plasmic space.
Each BtuC subunits traverses the
membrane 10 times
20 transmembrane helices in the
transporter.
The interface between the two BtuC
subunits is formed by anti-parallel
packing of two pairs of helices.
It has no structural resemblance to
the binding pockets of vitamin B12-
dependent enzyme.
24. Arrangement of ABC Domain
The BtuD subunits are
aligned such that two ATP
hydrolysis sites are formed
at the dimer interface
between the ABC signature
mofit and the P-loop of the
other.
Nucleotide binding and
hydrolysis is due to the
specific structure dimer
instead of an individual
domain.
Physiological dimers could
only be trapped under ATP-
EDTA condition.
BtuCs keep the BtuDs
properly aligned.
25. Transmission- BtuC-BtuD interface region
Architecturally conserved in ABC transporter
Q loop of BtuD involved in the interface with
BtuC.
Act as a alpha-phosphate sensor that may
change its conformation upon nucleotide
binding and hydrolysis.
When the binding protein BtuF has docked
with BtuC, it signals to the ATP hydrolysis
sites to initiate ATP hydrolysis.
The mutation of residues located at the
transmission interface either Interferes with
protein folding or assembly, or affects the
coupling of ATP hydrolysis to substrate
translocation.
For example, point mutation of Arg659 in
transporter severely affects coupling.
26. Importers have a high-affinity binding protein that specifically associates with the
substrate in the periplasm for delivery to the appropriate ABC transporter.
Importes
27. Exporters do not have the binding protein but have an intracellular domain that joins the
membrane-spanning helices and the ABC domain.
The ICD is believed to be responsible for communication between the TMD and NBD.
Exporters
29. Nucleotide binding domain (NBD) dimer interface is
held open by the TMDs and facing outward but
occluded from the cytoplasm.
Upon docking of the substrate-loaded binding protein
towards the periplasmic side of the transmembrane
domains, ATP binds and the NBD dimer closes.
This switches the resting state of transporter into an
outward-facing conformation, in which the TMDs
have reoriented to receive substrate from the binding
protein.
After hydrolysis of ATP, the NBD dimer opens and
substrate is released into the cytoplasm. Release of
ADP and Pi reverts the transporter into its resting
state.
31. The TMDs and NBDs are relatively far apart to accommodate
amphiphilic or hydrophobic substrates.
ATP binding induces NBD dimerization and formation of the
ATP sandwich, drives the conformational changes in the TMDs.
The cavity is lined with charged and polar residues that are
likely solvated creating an energetically unfavorable
environment for hydrophobic substrates and energetically
favorable for polar moieties in amphiphilic compounds or sugar
groups .
Since the lipid cannot be stable for a long time in the chamber
environment, lipid A and other hydrophobic molecules may
"flip" into an energetically more favorable position within the
outer membrane leaflet.
Repacking of the helices switches the conformation into an
outward-facing state.
32. In MDR, patients that are on medication eventually
develop resistance not only to the drug they are
taking but also to several different types of drugs.
This is caused by several factors, one of which is
increased excretion of the drug from the cell by ABC
transporters.
For example, the ABCB1 protein (P-glycoprotein)
functions in pumping tumor suppression drugs out of
the cell.
The most-studied member in ABCG family is ABCG2, also known as BCRP (breast
cancer resistance protein) confer resistance to most of Topoisomerase I or II inhibitors
such as topotecan, irinotecan, and doxorubicin.
Role in multidrug resistance
