What is the structure of ATP synthase?
ATP synthase is a multi-subunit enzyme.
• There are two main domains: FO and F1.
• FO causes rotation of F¬1 and is made of a c-ring and subunits a, b and d.
• The F¬1 domain can be detached from the membrane by using 8M of urea. It has a water-soluble part which can hydrolyse ATP. It is made up of α, β, γ, δ and ε.
• If c-ε-δ are cross linked, the enzyme will still function.
• Oligomycin sensitivity conferring factor is found in E. coli.
How can we study the rotation of ATP synthase?
ATP synthase has a rotating subunit and there are two ways in which we can study this.
Fluorescent filament
• This experiment showed that ATP hydrolysis drove rotation.
• His tags were added to β subunits in order to secure it to a nickel surface.
• Streptavidin was used to bind an actin filament to the c-ring.
• This experiment showed that there were three 120-degree rotations.
FRET
• C-ring and gamma subunit were labelled in E. coli.
• There were changes in FRET once a proton gradient was created in liposomes.
Metal beads
• Gold improved resolution and showed 90-degree and 30-degree steps in rotation.
• We can add a magnetic bead instead.
• The bead was added to the gamma subunit with streptavidin.
• The magnetic bead can drive rotation and lead to ATP synthesis.
How is the F1 subunit structured?
The F1 subunit is where ATP synthesis occurs.
• It has 5 subunits: α, β, γ, δ and ε. The stoichiometry is α3β3γδε.
• The γ subunit is 2 α helices with C and N termini in the α3β3 complex.
• α and β are very similar except in their N-terminal binding domains.
• β experiences a conformational change as the γ subunit rotates.
• α and β both have hydrophobic areas where the γ subunit can bind.
• Both subunits contain walker motifs (associated with phosphate binding).
• The γ subunit organises the α and β subunits. Rotation of it causes conformational changes in the β subunit.
• The δ subunit binds the F1 subunit to the membrane. It interacts with the α subunits of F1 and the b subunits of F1.
• The ε subunit is needed for F1 to correctly bind to FO. In bacteria and chloroplasts the subunit inhibits the activity of F1 so it has a regulatory role. In plants it makes synthase unidirectional. No ATP hydrolysis.
How does the β subunit undergo conformational changes?
The β subunits are involved in the actual conversion of ADP and phosphate into ATP. There are 3 distinct states which the subunits can be in. This is confirmed with crystal structures.
• Open: Nothing bound. Releases ATP.
• Loose: ADP and phosphate bind.
• Tight: The ADP and phosphate are bound so tightly that ATP is formed.
The mechanism causes these states to change.
• Rotation of γ by 120 degrees causes each β subunit to undergo a conformational change and adopt the next structure along.
• We can show this by labelling each subunit with a different colour.
• The interaction causing tis change is between a short 18 amino acid stretch of the α helix in the γ subunit and a conserved DELSEED motif on the β subunit. This interaction is seen in the ATP bound T state.
• The α subunit has a his-gly-gly motif near the N terminus.
How is the FO subunit structured?
The FO complex is made up of the a, b and c subunits.
• All 3 subunits are transmembrane.
• The c subunit contains the active site as it caries the proton.
• It consists of two transmembrane helices which are joined by a short loop. It protrudes out of the P phase of the membrane.
• DCCD inhibits proton movement by binding covalently to an acidic residue in the c subunit.
• Subunit b is found twice in an FO complex. It is found as part of the F1 stalk. There is a single transmembrane helix at the N-terminal end which makes up about 20% of the molecule. The rest of the molecule has 2 α helical sections which stretch out of the membrane and make contact with F1.
• Subunit a has a transmembrane helix which has a polar side which possibly interacts with the protonophore in subunit c forms part of the proton pore.
• Subunit a interacts with subunit b and transmits the effects of the c ring movement up to F1.
• Subunit b prevents α and β from rotating.
How does the FO subunit work thermodynamically?
ATP synthase is driven by ΔP.
• It can be driven by the Δψ, which is the electric potential gradient. This is the case in mitochondria and bacteria.
• It can also be driven by the pH gradient. This is the case in thylakoids.
• Critical amino acids of the C-ring are located about halfway through the membrane. Protons therefore experience a 50% drop in Δψ as they enter the IMS half channel.
• The proton gradient is seen by the FO rotor as a 3-pH unit gradient. This is regardless of whether the ΔP is dominated by either term.
How does the c-ring actually work? How is this different in mitochondria and bacteria?
Torque is the actual movement of the γ subunit itself.
• Protons are delivered via the IMS channel to E59 (mito) or D61 (E. coli).
• E59 is the only hydrophilic residue in the hydrophobic cylinder.
• Binding of protons compensates for loss of salt bridge with R176 (mitochondria) or R210 (E. coli).
• The ring rotates by one subunit per proton bound. Rotation means a protonated C subunit will move to the R residue.
• When the protonated carboxylic acid reaches the N half channel, the proton is unloaded.
• Loss of proton to the matrix half channel is compensated for by the electrostatic interaction gained by coming closer to the positive R.
• This sequence is repeated n times, where n is the number of c subunits.
Why do different organisms have different numbers of C-ring subunits?
The number of c-ring subunits relates to the proton/ATP ratio.
The higher the ratio, the greater the free energy that can be produced at a given ΔP at equilibrium.
Structural investigations can determine x’ as it is equal to the number of subunits divided by 3.
Enzymes with more c-subunits can therefore generate the same ΔG at a lower ΔP.
Plants will sometimes require ATP from a smaller gradient. Sometimes they will have weak light and need to make ATP.
