Module 0220502
Membrane Biogenesis and Transport
Lecture 12
Structure and Function of the
H+-Translocating ATP Synthase of
Energy-Coupling Membranes
Dale Sanders
26 February 2009
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 catalyse
H+ flow and ATP hydrolysis/synthesis, respectively;
• The fundamental subunit structure of each sector, and its
significance for H+ flow and ATP synthesis;
• The mechanism of ATP synthesis by rotational catalysis;
• The basic structure and function of Vacuolar H+-pumping
ATPases.
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 ATP
synthase. Arch. Biochem. Biophys. 476: 43-50.
Predicting Transmembrane Domains of Proteins with
Hydropathy Analysis
For most transport systems where 1° structure known, there are no
data on 2° and 3° structure. Therefore…
use computer algorithm to predict transmembrane spans on the basis
of 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 its
oil: 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 overall
hydropathy index >1
C
N
}
Hydropathy index
e.g. M subunit,
Rhodopseudomonas
Photosynthetic Reaction
Centre
+2.25
”windows” of 20 residues
calculate mean hydropathy
index
T/membr. spans
1.0
0
–2.25
0
50
100 150 200 250
Residue number
300
ATP Synthase of Energy Coupling
Membranes: A Protein of Central
Importance in Biology
Question: What weight of ATP does a 70 kg
human generate in a day?
Answer:
75 kg!!!!
H+ Translocation by the ATP Synthase of
Energy –Coupling Membranes: Basic Structure
ATP synthase is located on N side of membrane.
Can be visualised by negative staining or by cryo-EM
after 2D crystallization.
P
membrane
N
8 nm
4 nm
Direction of passive H+ flow
Cryo-EM of sub-mitochondrial particles
Properties of this macromolecular
complex in mitochondria:
Remove Ca2+ from solution and head-piece drops off.
Find large amount of solublized ATPase activity.
Importantly: In these conditions, membranes retain their
capacity 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 two
sectors of the enzyme have different roles in ATP
synthesis
Solubilized head-pieces catalysing ATP hydrolysis
can be added back to stripped smp’s (in presence of
Ca2+):
1. in presence of a PMF they synthesize ATP:
smp’s are coupled.
2. if resp. chain is blocked, and ATP is provided, the
whole complex pumps H+.
i.e.
(1)
(2)
Driving reaction in red
H+
ADP+Pi
H+
ATP
ADP+Pi
H+
H+
ATP
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)
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
Stoichiometry
Mr (k)
Disposition in membrane: evidence from
• hydropathy analysis
• models for globular proteins
• studies with interfacial reagents
• cryoelectron microscopy
a
1
30
b
2
17
c
10-14
8
C
N
D/E 61
P
C
N
a
N
C
N
b
c
Mechanism of H+ flow: AN INTERESTING FACT ABOUT F0:
•
D/E 61 on subunit c is essential
Covalently binds inhibitor dicyclohexylcarbodiimide
(DCCD)
Just 1 DCCD bound per holoenzyme is sufficient for
complete inhibition.
Implications for H+ Flow Through F0:
H+ translocation must involve all 10-14 c subunits.
F1 sector: Subunit Composition
subunit
stoichiometry
Mr (k )
α
β
γ
α
3
55
β
3
50
γ
1
31

1
20
ε
1
15
bind ATP tightly, but non-catalytic: function unknown
comprise catalytic binding sites for ATP
runs through centre of 3β3 hexamer
3β3 γ complex has been crystallized, and shows
alternating β array with γ in centre:

β

β


β
Also shows the 3 catalytic nucleotide-binding sites in different
states simultaneously on each β subunit
Open:
Nothing bound
Loose:
ADP + Pi bound
Tight:
ATP bound
Abrahams et al. (1994) Nature
370: 621-628
Abrahams et al. (1994) Nature
370: 621-628
Abrahams et al. (1994) Nature
370: 621-628
Abrahams et al. (1994) Nature
370: 621-628
How Does H+ Flow Through F0 Energise
ATP Synthesis by F1?
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
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)
The world’s smallest motor!!
 
Stator
membrane

b


Rotor
c ring
a
H+
Rotary Catalysis and Binding Site
Conformation in F1 – How ATP is Made
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.
ADP + Pi


Energy
ADP+Pi


P
AT
Pi
Pi
P+
P+
AD
AD
AT
P


ADP+Pi
ATP
Cross
(1994) Nature 370: 594-595
Note: Energy put into driving conformational changes in binding
sites especially in Opening the Tight site to get ATP off the surface
of the enzyme.
Stoichiometry: 4 H+/ATP = 12 H+ for full cycle.
H+ Flow and Rotary Catalysis
http://www.youtube.com/watch?v=uOoHKCMAUMc
Vacuolar ATPases (V-ATPases) are Distant
Cousins of F-ATPases
Functions: H+ pumping INTO the lumen of cellular
compartments e.g. lysosomes, Golgi, chromaffin granules, plant
and 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
Structure
2H+/ATP
Vo (= Fo) sector
V1 (= F1) sector
Many subunit types, amongst which…
in V0, a 16 kDa subunit
6 copies / holoenzyme
N
C
Both N & C halves homologous to subunit c of F0
Evolved from gene duplication and fusion
in V1,
70
Catalytic:
β homologue
& 60 kDa subunits
non-catalytic:
α homologue
3 copies each
SUMMARY
1.
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.