ABC transporters

A phospholipid bilayer acts as a selectively permeable barrier

A pure phospholipid membrane is permeable to

Gases: oxygen, carbon dioxide Hyrophobic molecules: benzene, glycerol Small uncharged polar molecules: EtOH All above molecules translocate in the process of passive diffusion

A pure phospholipid bilayer is impermeable to

Water soluble molecules Charged molecules such as ions Sugars Amino acids

How do these molecules enter a cell?

Membrane proteins form special passages through the membrane Pumps Carriers Channels

Membrane Transporter Proteins: Classification

Pumps
Primary active transporters Active = Use external energy Primary = they generate chemical or electrical gradients and later transport against the gradient Are enzymes (they have to break some bonds to produce energy) ATP driven pumps are also called ATPases Transport ions and small molecules Types of energy used by pumps ATP Light Redox potential Decarboxylation

Facilitated vs. passive dif fusion

ATP-binding cassette transporter

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.

ABC transport proteins

vincludes more than 200 different transport proteins v vEach ABC transport protein is specific for a single substrate v vMulti-domain structures that are comprised of two helical transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs).

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Black arrows represent sites of translocation by ABC transporters in Eukaryotic/Prokaryotic cells.

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

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STRUCTURE
vTMD (Transmembrane domain) v v vNBD (Nucleotide binding domain) v

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vMost transporters have transmembrane domains that consist of a total of 12 -helices with 6 -helices per monomer. v v vSince TMDs are structurally diverse, some transporters have varying number of helices (between six to eleven).

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Imp characteristics of these systems is that they share a highly conserved AT Pase domain v Binds v Hydrolyzes ATP Amino acid sequence of this cassette displays 3 major conserved motifs, the walker A and walker B motifs commonly found in ATPases together with a specific signature motif usually commencing LSGG also known as the linker peptide. Functions v Importers v Exporters v DNA repair, translation Regulation of gene expression

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NBDs of ABC Transporters are Defined by Five Sequences

ATP-binding motifs, P loop

Walker A GxxGxGKS(S/T)

Walker B hhhhDE

Conformational motifs

Glutamine Loop xxQx Signature Sequence LSGGQ

Histidine Loop hhhpH(+/-)

NBD 3-Dimensional Structure

His-loop

L-shaped structure
Signature Gln-loop Walker B Walker A ATP

cylinders are -helices arrows are -sheets ATP loosely bound by Walker A/B motifs

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The Q-Loop Mediate TMDNBD Communication A highly conserved glutamine residue

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ABC Tr anspor ter sare found in all living systems and have a common architecture: membrane integral subunits (green) that have a substrate binding site, with two ATP Binding Cassettes (pink) that power the system by binding and hydrolyzing ATP.

vNucleotide binding is required to ensure the electrostatic

& structural integrity of the active site. v Binding of ATP is stabilized by following interactions vRing stacking interaction of a conserved aromatic residue preceeding the WALKER A motif & the adenosine ring of ATP. vHydrogen bonds between a conserved lysineresidue in WALKER A motif & the Oxy atom of the & phosphates of ATP. v phosphates co-ordination with side chains of serine & backbone amide groups of Glycine residues in the LSGGQ motif. vIn addition a reside that suggests the tight coupling of ATP binding & dimerization is the conserved histidine residue in the H-loop. v

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vEnzymatic hydrolysis of ATP requires proper binding

of phosphates and positioning of phosphate to the attacking water. v In NB site, the Oxy atom of the & phosphates of ATP are stabilized by residues in the WALKER A motif & co-ordinate with Mg2+ ion. vThis Mg2+ ion also co-ordinates with the terminal aspartate residue in the WALKER B motif through the attacking water. v vGlutamine in the Q Loop or Histidine In the switch region that forms a hydrogen bond with phosphate of ATP is found to catalyze the rate of ATP hydrolysis by promoting the attacking water molecule. v

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ple ATP-switch mechanism power s ABC tr anspor

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This form of active transport involves both transporter proteins and the energy provided by the hydrolysis of ATP. A specific periplasmic-binding protein carries the substance to be transported to a membranespanning transporter.

The molecule to be transported across the membrane enters the transporter protein system and a molecule of ATP enters the ATP binding site of the ATP-hydrolyzing protein.

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Energy provided by the hydrolysis of ATP into ADP, phosphate, and energy moves the molecule across the membrane.

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The carrier protein releases the molecule being transported and the transporter system is ready to be used again.

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ABC systems of 3 types can be distinguished on the basis of design of their component parts Transporters 4 structural domains 2 hydrophobic membrane (IMS) domains 2 hydrophillic cytoplasmic domains Systems involved in cellular processes donot have IM domains and are composed of 2 ABC domains fused together.

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3 classes
Class 1 - systems with fused ABC and IM domains exporters Class 2 - systems with no known TM domains Ex:- antibiotic resistance, translation Class 3 - systems with TM and ABC domain s carried by independent polypeptide chains- importers

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Class1 (exporters) BAE Bacteriocin and peptide export MDL Mitochondrial peptide export HLY RTX toxine export PGP Eukoryote multiple drug rsistence and lipid export Class 2 RLI ART EF -3 REG UVR RNase L inhibitor Antibiotic resistance and translation regulation Translation elongation Translation regulation DNA repair and drug resistance

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Class 3 with unfused TM and ATP binding domain MET Metals OSP Oligosacharides and polyols Class 3 systems of unknown function that could be importers CBY Cobalt uptake Class 3 systems which are not known exporters 0228 lipoprotein release CDI Cell division Class 3 Systems which could be exporters DRA Drug and antibiotic resistence CLS Extracellular polysaccharide export NOS Nitrous oxide reduction

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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

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ABC TRANSPORTERS IN HUMNAS

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HUMAN ABC GENES

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A B C D E F G

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Commonly used drugs in chemotherapy

alkylating agents, anthracyclines prevent DNA replication vinca alkaloids, taxanes disrupt microtubule assembly etoposide inhibits topoisomerase II methotrexate prevents purine synthesis

ABC proteins affect drug distribution in tumour models

t accumulation of drug in tumour spheroid culture

non ABC expressing cells non ABC expressing cells

ABCB1/Pgp expressing cells

P-Glycoprotein

TM Domain

ATP binding

TM Domain

ATP binding

Pgp Substrates Adriamycin Daunorubicin Epirubicin Paclitaxel Docetaxel Vincristine Vinblastine VP-16 Mitoxantrone Actinomycin D

Pgp Inhibitors Verapamil Quinidine Quinine Cyclosporine A PSC 833 VX-710 LY335979 R101933 OC144-093 XR9576

Multiple ABC Transporters Confer Resistance to AntiCancer Drugs

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CONCLUSION

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ou ky an Th

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