Dale Sanders
26 February 2009
Module 0220502
Membrane Biogenesis and Transport
Lecture 12
Structure and Function of theH+-Translocating ATP Synthase of
Energy-Coupling Membranes
Aims:By the end of the lecture you should
understand…
• The significance of hydropathy analysis;
• That the F0 and F1 sectors of the ATP synthase catalyseH+ flow and ATP hydrolysis/synthesis, respectively;
• The fundamental subunit structure of each sector, and itssignificance for H+ flow and ATP synthesis;
• The mechanism of ATP synthesis by rotational catalysis;
• The basic structure and function of Vacuolar H+-pumpingATPases.
Reading
Lodish et al. (2004) Molecular Cell Biology pp. 326-9
is OK for the basics, but not very detailed.
Voet & Voet (2004) Biochemistry pp. 827-833
More detailed account:
• Nakamoto et al. (2008) The rotary mechanism of the ATPsynthase. Arch. Biochem. Biophys. 476: 43-50.
Predicting Transmembrane Domains of Proteins withHydropathy Analysis
For most transport systems where 1° structure known, there are nodata on 2° and 3° structure. Therefore…
use computer algorithm to predict transmembrane spans on the basisof dominantly hydrophobic character: Hydropathy Analysis
Principles:
1. Hydrophobic polypeptide in hydrophopic environment adopts -helical conformation.
2. Hydrophobic span of bilayer 3 nm (30Å)
3. 3 nm of -helix 20 residues.
4. Assign a hydropathy index to each amino acid based on itsoil: water partition coefficient
values range from: + 4.5 (most hydrophobic: Ile)to – 4.5 (most hydrophilic: Arg)
5. Search sequence for stretches of 20 residues which have overallhydropathy index >1
+2.25
1.0
0
–2.250 50 100 150 200 250 300
Residue number
Hyd
rop
ath
yin
de
x
} ”windows” of 20 residues
calculate mean hydropathyindex
N C
e.g. M subunit,RhodopseudomonasPhotosynthetic ReactionCentre
T/membr. spans
ATP Synthase of Energy CouplingMembranes: A Protein of Central
Importance in Biology
Question: What weight of ATP does a 70 kghuman generate in a day?
Answer: 75 kg!!!!
H+ Translocation by the ATP Synthase ofEnergy –Coupling Membranes: Basic Structure
ATP synthase is located on N side of membrane.Can be visualised by negative staining or by cryo-EMafter 2D crystallization.
membrane
8 nm
4 nm
P N
Direction of passive H+ flow
Cryo-EM of sub-mitochondrial particles
Properties of this macromolecularcomplex in mitochondria:
Remove Ca2+ from solution and head-piece drops off.
Find large amount of solublized ATPase activity.
Importantly: In these conditions, membranes retain theircapacity for electron transport after removal of head-piece.
They are uncoupled: - respiratory rate increases
- membrane leaky to H+
Function:
From these results we can conclude that the twosectors of the enzyme have different roles in ATPsynthesis
Solubilized head-pieces catalysing ATP hydrolysiscan be added back to stripped smp’s (in presence ofCa2+):
1. in presence of a PMF they synthesize ATP:smp’s are coupled.
2. if resp. chain is blocked, and ATP is provided, thewhole complex pumps H+.
Conclusions:
The ATPase is REVERSIBLE: a pump or a synthase
The head-piece is involved in ATP synthesis/hydrolysis
The head-piece is called F1
The stalk forms a H+ channel, which is open in the absence of F1.Stalk is called Fo
Generically known as F-TYPE ATPases
Present on all energy-coupling membranes (mitos, thylakoids,prokaryote)
H+
H+
ADP+Pi
ATP
H+
H+
ADP+Pi
ATP
i.e. (1) (2)Driving reaction in red
Structure and Function of Subunits
Most work on E. coli enzyme which has fewest sub-unit types:Encoded on unc operon Mr = 540 k
F0 sector Subunit a b c
Stoichiometry 1 2 10-14
Mr (k) 30 17 8
Disposition in membrane: evidence from
• hydropathy analysis
• models for globular proteins
• studies with interfacial reagents
• cryoelectron microscopy
a b cMechanism of H+ flow: AN INTERESTING FACT ABOUT F0:
• D/E 61 on subunit c is essentialCovalently binds inhibitor dicyclohexylcarbodiimide(DCCD)Just 1 DCCD bound per holoenzyme is sufficient forcomplete inhibition.
Implications for H+ Flow Through F0:H+ translocation must involve all 10-14 c subunits.
D/E 61
N C N CN
C
P
N
F1 sector: Subunit Composition
subunit α β γ ε
stoichiometry 3 3 1 1 1
Mr (k ) 55 50 31 20 15
α bind ATP tightly, but non-catalytic: function unknown
β comprise catalytic binding sites for ATP
γ runs through centre of 3β3 hexamer
3β3 γ complex has been crystallized, and showsalternating β array with γ in centre:
Also shows the 3 catalytic nucleotide-binding sites in differentstates simultaneously on each β subunit
Open: Nothing bound
Loose: ADP + Pi bound
Tight: ATP bound
β
β
β
Abrahams et al. (1994) Nature370: 621-628
Abrahams et al. (1994) Nature370: 621-628
Abrahams et al. (1994) Nature370: 621-628
Abrahams et al. (1994) Nature370: 621-628
Putting together kinetic and structural data, the model
of rotational catalysis has been developed:
1. H+ flows passively through channels provided
jointly by subunit a and 1 of c subunits.
2. Movement of H+ drives rotation of a ring of c
subunits
[Recall: 1 DCCD bound inhibits catalysis completely]
3. γ is connected indirectly (via ε) to c ring, and also
rotates
How Does H+ Flow Through F0 EnergiseATP Synthesis by F1?
a
b
c ring
H+
membrane
The world’s smallest motor!!
5. Subunits a, α and β are prevented from moving by
subunits b (a “stator”)
6. Rotation of γ drives each of catalytic sites through
conformational change (O L T)
Stator Rotor
1. ADP + Pi bind freely to Loose binding site.
2. Rotation of γ conformational change,
making the Loose site Tight.
3. In the Tight site ATP forms spontaneously.
4. The Tight site Opens and ATP is released,
again as γ rotates.
Rotary Catalysis and Binding SiteConformation in F1 – How ATP is Made
Note: Energy put into driving conformational changes in bindingsites especially in Opening the Tight site to get ATP off the surfaceof the enzyme.
Stoichiometry: 4 H+/ATP = 12 H+ for full cycle.
ADP + Pi
AD
P+P
i
ATP
ADP+Pi
AD
P+P
i
ADP+Pi
ATP
ATP
Energy
Cross (1994) Nature 370: 594-595
http://www.youtube.com/watch?v=uOoHKCMAUMc
H+ Flow and Rotary Catalysis
Vacuolar ATPases (V-ATPases) are DistantCousins of F-ATPases
Functions: H+ pumping INTO the lumen of cellularcompartments e.g. lysosomes, Golgi, chromaffin granules, plantand fungal Vacuoles
Physiological roles: H+ - coupled solute accumulation
vesicle trafficking
Also H+ pumping OUT of a few cell types
e.g. osteoclasts – Bone resorption
intercalated cells of renal collecting tubule -Urinary acidification
Stoichiometry 2H+/ATP
Structure Vo (= Fo) sector
V1 (= F1) sector
Both N & C halves homologous to subunit c of F0
Evolved from gene duplication and fusion
in V1, 70 & 60 kDa subunits 3 copies each
Catalytic: non-catalytic:
β homologue α homologue
Many subunit types, amongst which…
in V0, a 16 kDa subunit
N C
6 copies / holoenzyme
SUMMARY1. Hydropathy analysis predicts transmembrane spans in
sequences of membrane proteins.
2. ATP synthase composed of 2 sectors:
Fo F1
H+-conducting ATP binding
3 subunit types 5 subunit types
3. ATP is synthesized by ROTARY CATALYSIS
H+ flow through Fo drives rotation of subunits and
conformational energy is transmitted to F1 driving each
binding site through a series of affinity changes.
4. Vacuolar H+-ATPases in organelles are distantly related to F
– ATPases – Function solely as PUMPS.
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