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.