Download ATP-driven Pumps

Module 0220502
Membrane Biogenesis and Transport
Lecture 13
ATP-driven Pumps
Dale Sanders
2 March 2009
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
+
– ATP
+ –
3Na+/2K+/ATP :
3Na+
ADP + Pi
–
An electrogenic pump
2K+
+
• 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.
A “housekeeping” enzyme
Synergistic stimulation of ATPase activity by Na+
and K+ in animal plasma membranes:
ATPase activity
Na+ = 40 mM
Na+ = 10 mM
Na+ = 0
[K] / mM
120
2. Fungal, plant H+-ATPase
pH 7
ATP
+
H
ADP + Pi
Note: 1H+ /ATP
pH 5 or lower
•
expels excess H+ produced during metabolism:
cytosolic pH regulation
•
maintains PMF: H+ gradient used to energize
transport
3. Sarcoplasmic Reticulum Ca2+ - ATPase
[also on ER, hence “SERCA” pump]
Note: an intracellular
membrane
myofibrils
SR
sarcoplasm
mM
nM Ca2+
2Ca2+
ADP + Pi
2H+
ATP
• Sequesters Ca2+ in relaxed muscle
4. Plasma membrane Ca2+-ATPase
Ca2+
ATP
nM Ca2+
ADP + Pi
mM Ca2+
•
Ubiquitous in
eukaryotes
2H+
In vivo activity ~ 5%
of (Na+/K+)-ATPase
Maintains low [Ca2+]cyt in order to…
…eliminate Ca2+ toxicity
…serve as baseline for amplification during signalling
•
Ca2+ activation through calmodulin binding domain
5. Gastric mucosal H+/K+-ATPase
stomach
K+
H+
lumen
ATP
H+
blood
ADP + Pi
pH 7.5
pH 1.0
• Unlike other pumps, this is electroneutral, not electrogenic
•
Acidification of stomach lumen
•
A target for proton pump inhibitors used to counter diseases associated
with stomach acidity e.g. dyspepsia, stomach ulcers
Omeprazole, an H/K-ATPase inhibitor
6. E coli K+ uptake system: Kdp-ATPase
ATP
+
K
ADP + Pi
•
A bacterial example
•
Upregulated at low external [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:
K+
Na+, ATP
IN
OUT
ADP
The “Post-Albers” scheme
(K)E1
E1-P(Na)
(K)E2
E2-P(Na)
Pi
K+
Na+
[compare F- & V-type ATPases, where no covalent
phosphorylation]
H2VO4– competes with Pi for binding; stabilizes transition state
For this enzyme, E1 is Na+- and ATP-binding [Ca2+ binding for
Ca2+-ATPases]
E2 is K+-binding
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:
cytosolic side
N
T
G
T
T K
G D*
E
S
G
D
G
X
N
D
* the phosphorylated asp residue
C
only 4% on
extracytosolic side
SR Ca2+-ATPase at 2.6 Å resolution – Ca2+ binding state
(E1)
80 Å
(8 nm)
A domain
contains
conserved
TGES
motif
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
C
X
X
C
C
X
X
C
C
X
X
C
C
X
X
C
C
X
X
C
C
X
X
C
T
G
E
S
T
G
T
K
D
N
G
D
G
X
N
D
C
X
P
C
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
Analysing CPx Transporter Function:
Complementation of a Yeast Mutant Defective in Cd2+ Tolerance
By an Arabidopsis CPx Transporter (AtHMA4)
Yeast mutant (ycf1)
Wild type
Control
AtHMA4-expressing
Mills et al. (2005) FEBS Lett. 579: 783
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 on
heavy 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
Solute binding protein
ATP
Small solute (eg his)
Membranebound ATPase
component
Inner membrane (tight)
Outer membrane (permeable to
small solutes)
System distinct from F-, V-, + P-type 1º pumps
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
Human
RING 4-11
Peptides
Out
Into E.R.
ABC Transporters Share a Common Domain Structure
Binding protein
(Gram –ve bacteria only)
Tramsmembrane Domain (TMD)
Membrane
Encoded:
6 t/m
spans
A
C
ATP
binding
6 t/m
spans
B
D
ATP
binding
Nucleotide-binding Domain
(NBD)
all on separate genes (esp. prokaryotes)
or fused pairwise …. either AB & CD or AC & BD
or all one 1 gene (eg MDR1, CFTR)
A dimer: 2 x (TMD + NBD)
Membrane chamber,
open extracellularly
Intracellular loops
ADP
From intracellular side
From extracellular side
Higgins (2007) Nature 446: 749
Membrane
Structure of a Bacterial ABC Multidrug-Transporter
(Sav1866 from Staphylococcus aureus)
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.