ATP Synthase

Module 711: Molecular Machines

Lecture 6

Dale Sanders

25 November 2009

Aims:By the end of the lecture you should understand…

• The structures and roles of the subunits;

• What the binding change hypothesis is, and theevidence for it;

• The molecular basis for the binding changehypothesis in terms of subunit interactions;

• How protons flow through the F0 sector;

• Rotational catalysis.

ReadingEssential reviews:• Stock et al. (2000) The rotary mechanism of ATP synthase. Curr.Opin. Struct. Biol. 10: 672-679;

• Senior et al. (2002) The molecular mechanism of ATP synthesisby F1F0-ATP synthase. Biochim. Biophys. Acta 1553: 188-211.

• Weber & Senior (2003) ATP synthesis driven by proton transportin F1F0-ATP synthase. FEBS Lett. 545: 61-70.

• Walker & Dickson (2006) The peripheral stalk of the mitochondrialATP synthase. Biochim. Biophys. Acta 1757: 286-296

• Nakamoto et al. (2008) The rotary mechanism of the ATPsynthase. Arch. Biochem. Biophys. 476: 43-50

• Ballmoos et al. (2009) Essentials for ATP synthesis by F1F0 ATPsynthases Annu Rev Biochem. 78: 649-672

More detailed research papers are referred to in reviews

ATP:The central energy-transducing molecule

• A 70 kg human generates about 75 kg per day;

• Used by animals, plants and microbes alike;

• Most is produced at energy-coupling membranes:bacterial plasma membrane

thylakoid membrane

mitochondrial inner membrane

• Driving force for ATP synthesis: theprotonmotive force.

PMF [C = 59 mV]

ATP Synthase:The membrane-located enzyme catalysing PMF-driven

ATP synthesis

• Present on all energy-coupling membranes

• Comprises two multi-subunit sectors: F0, F1;

• F0 is membrane-integral and catalyses passiveproton flow;

• F1 is non-integral, located on low-proton-potential side and when detached catalyses ATPhydrolysis;

A Current View of the F-ATPase

Ballmoos et al. (2009)Annu Rev Biochem. 78: 649-672

Cryo-EM of sub-mitochondrial particles

Subunit composition:The E.coli enzyme has the minimum number of subunit

types

• F1

subunit α β γ ε

stoichiometry 3 3 1 1 1

Mr (k) 55 50 31 19 15

• F0

subunit a b c

stoichiometry 1 2 10-15

Mr (k) 30 17 8.3

t/membr. spans 6 1 2

Total Mr = 544 k

Cryo-EM image of E. coli complex showingtwo “stalks”

F1

sector

F0

sector

stalk stalk

Interpretation of EM image at subunit level

The structure of F1

33 complexcrystallizedfrom beef heartmitochondriaand resolved at2.8 Å byAbrahams et al(1994) Nature 370:621

20 Å

Structure of F1:

• Crystallised in presence of ADP and ATPanalogue, AMP-PNP (non-hydrolysableimidodiphosphate bond);

• Most of and resolved but < ½ ;

• and alternate around “rod”;

• 3 subunits each bind AMP-PNP;

• subunits contain either– AMP-PNP (TP)

– ADP (DP)

– Nothing (E)

• subunit: 2 helices forming coiled coil.

“Jaw”swings openwhen siteempty

Asymmetry of subunit

Interaction with and at “top” through hydrophobicresidues at C and N termini: a “greased bearing”

View from membrane side

Horizontal slice across top showing sheetstructure that provides cap across nucleotide

binding domains

20 Å

Horizontal slice across NB domain, primarilyhelical

http://nature.berkeley.edu/~hongwang/Project/ATP_synthase/

Side view Cutaway view inplane of membrane

Subunit b

F6

Subunit d

The Peripheral Stalk from BovineMitochondrial F-ATPase

Dickson et al. (2006) EMBO J 25: 2911

5 nm

Binding changes and cooperativity

Observations:

1. 18O exchange measurements showed that ATP ADP + Pi at equilibrium on enzyme

2. ATP found to promote release of tightly-boundADP

3. Stopped flow kinetic experiments foundnegative cooperativity for binding affinity, positivecooperativity for Vmax.

Binding changes and cooperativity

Catalytic mode Kd, ATP (M) Vmax (s-1)

Unisite 10-12 10-4

Bisite 3.10-5 300

Trisite 2.10-4 600

Conclude:

ATP hydrolysis is close to equilibrium on surface of enzyme.

Nucleotide binding is cooperative, with each site undergoingsequential affinity change.

Rotation within F1 drives affinity changesin NB sites essential for catalysis:

• The rod rotates, driving each of the NB siteson through changes in nucleotide affinity;

• Affinity changes allow sequential binding anddissociation of nucleotides to each site

• This rotation demonstrated by

Biochemical methods

Direct visualisation

Substitute Cys at critical residues in (e.g. D380C) and (e.g. S87C)

1. Oxidise to form S-S

2. Dissociate, mix with35S-labeled or :spontaneousreassociation

3. Reduce inpresence ofMgATP: findnew bridgesbtw unlabelled and labelled

Biochemical demonstration of rotation

Direct visualisation of rotation

1. Mount F1 at “top” on Ni-coated glass slide with His-tag a N terminus of

2. Engineer (S107C) and (C193S) to enable specific reaction of with biotin

3. React complex with biotinylated fluorescent actin probe with streptavidin linker

4. Actin filament turns counterclockwise when provided with MgATP

5. At very low [MgATP], discrete 120º transitions visualised.

High-speed imaging reveals Pi release inlast 40o of 120o

Adachi et al., (2007) Cell 130: 309

Rotation observed using goldbeads: reduces frictional drag

Two Models for Pi Release

Protein-protein interactions

Determined through random and site-directed mutagenesis;Second site suppressor mutationsInspection of crystal structure

• R268 and Q269 H-bonds with D303,T305 and D306

• R242 H-bonds with E381 in conserved380DELSEED386 sequence

Interpretation: rotary catalysis

Energy put into…

1. Lowering affinity for ATP

2. Raising affinity for Pi

a b c

• D/E 61 on subunit c is essentialCovalently binds inhibitordicyclohexylcarbodiimide (DCCD)Just 1 DCCD bound per holoenzyme issufficient for complete inhibition.

D/E 61

N C N CN

C

P

Role of F0 cytoplasm

Subunit-subunit interactions: F1 and F0

Residues on c subunit exhibiting second-sitesuppression of E208K

Generation of rotation

A Modelled View of Interactions BetweenSubunits a and c

Ballmoos et al. (2009)Annu Rev Biochem. 78: 649-672

Based on the Ilyobacter tartaricus F-ATPase

Model for H+ Transport Through F0

+ = aArg

- = cGlu/Asp

The H+/ATP Ratio

• The -subunit rotates through 120˚ per ATP made

• 9 c-subunits/ring fits 3H+/ATP

• 12 c-subunits/ring fits 4H+/ATP

• 10 c-subunits implies non-integral H+/ATP

• 14 c-subunits in chloroplasts

• 11 c-subunits in Na+-motive ATPases

• Most estimates of H+/ATP are between 3 and 4

SummaryWe have covered…

• The structures and roles of the subunits;

• What the binding change hypothesis is, and theevidence for it;

• The molecular basis for the binding changehypothesis in terms of subunit interactions;

• How protons might flow through the F0 sector;

• Rotational catalysis.