Dale Sanders

2 March 2009

Module 0220502

Membrane Biogenesis and Transport

Lecture 13

ATP-driven Pumps

Aims:By the end of the lecture you should

understand…

• The distinguishing features and physiological

importance of P-type ATPases;

• The way in which P-ATPase structure is related

to mechanisms of ion translocation;

• What CPx ATPases are, and how they are related

to other P-type ATPases;

• The structural attributes and significance of ABC

transporters.

Reading Lodish et al (2004) Molecular Cell Biol 5th ed, pp.252-7

A good, brief, introduction to P-type ATPases.

More detailed accounts:

• Toyoshima et al. (2000) Nature 405: 647

• Olesen et al. (2007) Nature 450: 1036

papers on SR Ca2+-ATPase structure

• Morth et al. (2007) Nature 450: 1043

paper on Na+,K+-ATPase structure

• Solioz & Vulpe (1996) Trend. Biochem. Sci. 21: 237

article on CPx ATPases

• Higgins (2007) Nature 446: 749

ABC transporters and multidrug resistance

P-Type ATPases: A Widespread Family with

Fundamental Physiological Roles

UNIFYING FEATURES:

• Single large catalytic monomer, 70 – 200 kDa

• Inhibition by μM orthovanadate, H2VO4-

• ATP donates γ- to conserved Asp residue during catalysis

• all pump irreversibily

• all pump cations

What do they do? Some Examples…

1.(Na+/K+) - ATPase of animal cell plasma membranes

• Maintains K+-rich cytosol – essential for protein synthesis etc.

- probably originally its primary function

• Maintains a Na+-motive force: used to energize coupled transport.

• Both Na+ and K+ gradient exploited during electrical signalling.

ATP

ADP + Pi

3Na+

2K+

+

+

+

–

– –

3Na+/2K+/ATP :

An electrogenic pump

AT

Pa

se

activity

[K] / mM

Na+ = 40 mM

Na+ = 0

Na+ = 10 mM

120

A “housekeeping” enzyme

Synergistic stimulation of ATPase activity by Na+

and K+ in animal plasma membranes:

• expels excess H+ produced during metabolism:

cytosolic pH regulation

• maintains PMF: H+ gradient used to energize

transport

2. Fungal, plant H+-ATPase

pH 7H+

pH 5 or lower

ATP

ADP + Pi

Note: 1H+ /ATP

3. Sarcoplasmic Reticulum Ca2+ - ATPase

[also on ER, hence “SERCA” pump]

• Sequesters Ca2+ in relaxed muscle

mM

2H+

ADP + Pi

Note: an intracellular

membrane

ATPnM Ca2+

myofibrils

SR

sarcoplasm

2Ca2+

4. Plasma membrane Ca2+-ATPase

• Maintains low [Ca2+]cyt in order to…

…eliminate Ca2+ toxicity

…serve as baseline for amplification during signalling

• Ca2+ activation through calmodulin binding domain

2H+ADP + Pi

Ubiquitous in

eukaryotesATP

mM Ca2+

Ca2+

In vivo activity ~ 5%

of (Na+/K+)-ATPase

nM Ca2+

pH 7.5

H+

ADP + Pi

ATPK+

• Acidification of stomach lumen

• A target for proton pump inhibitors used to counter diseases associated

with stomach acidity e.g. dyspepsia, stomach ulcers

bloodH+

pH 1.0

stomach

lumen

• Unlike other pumps, this is electroneutral, not electrogenic

5. Gastric mucosal H+/K+-ATPase

Omeprazole, an H/K-ATPase inhibitor

6. E coli K+ uptake system: Kdp-ATPase

• A bacterial example

• Upregulated at low external [K+]

ADP + Pi

ATP

K+Net cation import

Biochemical Properties: The Na+/K+ ATPase

• Trypsin cleavage sites are differentially exposed depending on

presence of Na+ or K+:

ion-dependent conformational changes

100 kDa

N C

+ Na+ + K+ trypsin cleavage sites

• The phosphorylated (E-P) intermediate is stabilized by Na+ and ATP

in the absence of K+:

E-P intermediate is discharged by K+

Mechanism of Transport

Results have given rise to the “E1E2” model for catalysis and transport:

Na+, ATPK+

ADP

Pi

(K)E1

(K)E2

K+

IN

OUT

E1-P(Na)

E2-P(Na)

Na+

[compare F- & V-type ATPases, where no covalentphosphorylation]

H2VO4– competes with Pi for binding; stabilizes transition state

For this enzyme, E1 is Na+- and ATP-binding [Ca2+ binding forCa2+-ATPases]E2 is K+-binding

The “Post-Albers” scheme

Significance of Conformational States

“E1 & E2” conformational states are associated with

binding site orientation: ATP hydrolysis

drives alternation in binding site orientation

In addition there is a change in binding site

affinity:

allows ion pick-up at low concn on one side

allows ion release at high concn on other side

These are the two ways in which free energy from

ATP hydrolysis is expended.

Structure of P-ATPases

Hydropathy analysis:

TGTKD*

TGES

GDGXNDN C

only 4% onextracytosolic side

cytosolic side

* the phosphorylated asp residue

SR Ca2+-ATPase at 2.6 Å resolution – Ca2+ binding state

80 Å

(8 nm)

(E1)

A domaincontainsconservedTGESmotif

MacLennan & Green (2000)Nature 405: 633

Transport

Mechanism of Ca2+-

ATPase

Comparing this structure with

that in Ca2+- free (E2) state……

1. Kinase activity unleashes N domain

from P domain

2. A domain associates with N & P

domains, exerts downward push on

M3/M4, opening luminal pathway for

Ca2+ release

3. ATP binding prevents reversal,

TGES triggers dephosphorylation

4. Cytoplasmic pathway opens for

Ca2+binding

Olesen et al. (2007) Nature 450: 1036

CPx – ATPases are P-TYPE ATPases that

Transport Heavy Metals

Examples:

Cd2+ export: in many bacteria, plants

Cu+ import: bacterial, human intestine*

Cu+ export: toxic levels disposed of in

bacteria, yeast, human

* Defect Cu deficiency in brain: Menkes

and Wilson diseases

Structure of CPx ATPases

N

Note: N terminal extension

CXXC motif found in other metal (eg Hg2+) binding proteins

Intramembrane CPx (= CPC, CPH or CPS) motif conserved among

all this subclass

Absence of extensive transmembrane spans at C terminus

TGES

TGTKD

GDGXND C

CXXC

CXXC

CXXC

CXXC

CXXC

CXXC

XPC

Analysing CPx Transporter Function:

Complementation of a Yeast Mutant Defective in Cd2+ Tolerance

By an Arabidopsis CPx Transporter (AtHMA4)

Mills et al. (2005) FEBS Lett. 579: 783

Yeast mutant (ycf1)

Wild type

Control AtHMA4-expressing

HMA4 is a key to tolerating heavy metals in plants (Hanikenne et al. (2008) Nature 453:391)

Constitutive overexpression of HMA4 in Arabidopsis halleri facilitates growth onheavy metals.

ABC Transporters

Ubiquitous: a diverse class, a superfamily, unified by

presence of ATP Binding Cassette in 1º structure:

GX(S,T)GXGK(S,T)(S,T)

Initially discovered as

Binding protein-dependent uptake systems that are:

Exclusive to Gram -ve bacteria

Sensitive to osmotic shock:

lose capacity for uptake of small solutes (e.g.

histidine, glutamine, arginine) due to release of

binding proteins in periplasmic space.

In bacteria, binding proteins can mediate

interaction between solute and ATP-dependent

transport system in inner membrane

System distinct from F-, V-, + P-type 1º pumps

ATP

Solute binding protein

Small solute (eg his)

Inner membrane (tight)

Outer membrane (permeable tosmall solutes)

Membrane-bound ATPase

component

ABC Transporters Translocate a Wide Variety of Solutes

Some examples:

Species System Substrate Direction

Streptococcus pneumoniae AmiABCDEF Oligopeptides In

E coli HisJQMP Histidine In

E coli PstABC Phosphate In

Erwinia chrysanthemi PrtD Proteases Out

Yeast STE6 a-mating peptide Out

Human MDR1 Hydrophobic drugs Out

Human CFTR Chloride Out

Human RING 4-11 Peptides Into E.R.

ABC Transporters Share a Common Domain Structure

Encoded: all on separate genes (esp. prokaryotes)

or fused pairwise …. either AB & CD or AC & BD

or all one 1 gene (eg MDR1, CFTR)

6 t/mspans

ATPbinding

6 t/mspans

ATPbinding

A B

C D

Binding protein(Gram –ve bacteria only)

Membrane

Tramsmembrane Domain (TMD)

Nucleotide-binding Domain(NBD)

Structure of a Bacterial ABC Multidrug-Transporter

(Sav1866 from Staphylococcus aureus)

A dimer: 2 x (TMD + NBD)

Intracellular loops

ADP

Membrane chamber,

open extracellularly Me

mb

ran

e

From intracellular side

From extracellular side

Higgins (2007) Nature 446: 749

Some Substrates of Multidrug Transporters

• Heterocyclic

• Lipophilic

• Mr < 800

Clinical Implications – and Mechanistic

ones too

MDRI: “Multidrug Resistance” – “P-glycoprotein”

Over-expressed in cells resistant to chemotherapy

Responsible for cytosolic clearance of a range of drugs

CFTR: “Cystic Fibrosis Transmembrane–

conductance Regulator”

The CI- channel defective in CF maps to this ABC-type transporter

Unusual to have a channel with the structure of a pump!

Summary

1. “P-type” ATPases are major cation pumps, performing a

variety of functions.

2. They form a phosphorylated intermediate and undergo

discrete conformational changes during the catalytic cycle.

3. The conformational changes are associated with (a) binding

site reorientation and (b) binding site affinity changes.

4. CPx–ATPases are a distinct sub-class of P-ATPase, and

pump heavy metals.

5. ABC transporters pump a wide variety of solutes and are

characterised by two distinct Nucleotide-Binding Domains.