ABC ORATP BINDING CASSETTE

TRANSPORTER

BY JASKAMAL GILLM-PHARM (PHARMACOLOGY)2nd semester

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

MECHANISMS INVOLVED IN MDR

Enzymatic degradation

Mutation at binding site

Down regulation of outer membrane proteins

Efflux pumps

Transduction of genes

Enzymatic degradation Mechanisms of b-lactamase

N

ON

O

OH

S CH3

CH3O

RH

b-lactamase

CH2

OHb-lactamase

CH2

OH

N

ON

O

OH

S CH3

CH3O

RH

b-lactamase

CH2

OH H2O

N

ON

O

OH

S CH3

CH3O

RH

HOH

b-lactamase

CH2

OH

+Hydrolysis of Oxyimino group

Penicillin drug

Inactivated drug

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

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).

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.

Multiple Drug Resistance

Proteins or ABC Proteins

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

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

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

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.

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

LONG & SHORT MRPs

Some Properties of the Human MRP family

04/12/2023 17Transporters

Structure

of

ABC

Transporters

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)

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)

BtuCDF complex structure and transport mechaism

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

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• the transmission interface.

Translocation Pathway of BtuCDPresent 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.

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.

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.

Importers have a high-affinity binding protein that specifically associates with the substrate in the periplasm for delivery to the appropriate ABC transporter.

Importes

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

Mechanism of transport for importers

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.

Mechanism of transport for exporters

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.

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

THANKS FOR CO-OPERATION

JASKAMAL GILL