Molecular Architecture of the Rotary Motor in ATP Synthase

Ahmed Ibrahim

Structure and Function Relationships of Membrane Proteins

November 14, 2006

Daniela Stock, Andrew G. W. Leslie, John E. Walker

Perspective

1. Adenosine triphosphate (ATP) is the principle unit of energy for most, if not all biological systems

2. The primary source of ATP in eukaryotic systems is the mitochondria (95%)

3. ATP is generated by catalyzing the addition of an inorganic phosphate to adenosine diphosphate

4. ATP is synthesized by enzymes in the mitochondria called ATP synthases

1937 1961 1964 1981 1994 1997

Herman Kalckar (Denmark) links ATP synthase is linked with

cell respiration

The American Ephraim Racker isolates the F1 part of the ATP synthase.

Peter Mitchell (UK) shows that cell respiration leads to pH change inside and outside the mitochondrial membrane

John E. Walker determines the DNA sequence of the genes encoding the proteins in ATP synthase.

Walker and coworkers determine The structure of the F1 part of the ATP synthase

ATP synthase rotate during the synthesis and hydrolysis of ATP is demonstrated by Richard Cross (USA) Wolfgang Junge (Germany) and Masasuke Yoshida (Japan)

1. ATP Synthase: a general term for enzymes that synthesizes adenosine triphosophate (ATP)

2. Conserved across species (earliest form found in archaea

3. In most systems it is embedded in a lipid bilayer and catalyzes the synthesis of ATP from adenosine diphosphate and an inorganic phosphate.

4. Fueled by proton motive force established by other membrane bound enzymes of the respiratory chain.

5. Completely Reversible

ADP + Pi + nH+P ATP + nH+

N

P: positive side of the membrane (mitochondrial matrix)

N: Negative side of the membrane (inner mitochondrial membrane)

Perspective

ATP Synthase Composition

1. Composed of 100,000 atoms

2. The primary source of ATP in eukaryotic systems is the mitochondria (95%)

3. ATP is generated by catalyzing the addition of an inorganic phosphate to adenosine diphosphate

4. ATP is synthesized by enzymes in the mitochondria called ATP synthases

Solvent exposed F1 Complex

Membrane embedded F0 Complex

Central Connecting Stalk gamma

3α 3β:1γ:1δ:1ε. (ATPase Activity)

1γ

a, b, and c subunits 1:2:9-12

F1 Synthase Composition

1. Catalytic sites are at the β subunits at the α/β interface

2. The γ subunit protrudes from the α3β3 and crosslinks with

the polar loop subunits of the c domain of the F0 subunit.

3. Rotation of the γ subunit is generated by the passage of protons through F0

4. Because there is only one asymmetric γ subunit, it interacts with different affinities with each of the three catalytic β subunits in F1 giving them different nucleotide affinities.

5. BINDING CHANGE MECHANISMS

With each rotation each one of the subunits transit through structural states: low (release of ATP), medium (binding of ATP and Pi), or high affinity (ATP formation)

120° goes through one of three subunit pair (full circle)

Challenge

1. F1 is very well defined through studies from bovine systems

2. Little is known about the structural details of the F0 subunit

1. All species contain three domains in the F0 subunit: a, b, and c

2. In E. coli the stoichiometry of these subunits is a1:b2:c9-11

3. Cross-linking and genetic experiments have shown that there are 12 c subunits per F0

4. Biochemical and mutational experiments have shown that the a and c subunits contain functional groups that are necessary for proton translocation through the membrane.

6. Nuclear Magnetic Resonance (NMR) has shown the structure of a c subunit as two alpha-helices linked by an extra-membrane loops.

Current Data on F0 of ATP Synthase Composition

Nuclear Magnetic Resonance (NMR)-derived Structure of the c Subunit of the F0 Portion of ATP Synthase

Mutational (truncation) studies in Saccharomyces cerevisiae have shown that Asp61 in E. coli and Glu59 at the COOH end of the c subunit is necessary for proton translocation

Proposed Models and Data for F0 Architecture

1. The a & b subunits align the exterior of the multimeric c subunits

2. The cavity in between a and c subunits providing the pathway for protons

3. Protonation and deprotonation in this interface is thought to cause rotation of the c ring thereby rotating the γ subunit.

4. The synthesis of ATP requires 120 degrees.

5. In certain some synthases with a 9 c subunit ring require 3 proton translocations

6. In other synthases with a 10 c subunit ring require 4 proton translocations

Proposed Models and Data for F0 Architecture

1. Crosslinking experiments and studies proposed that the b subunits of F0 act as a peripheral stator to keep the αβ subunits from rotating along with the γ subunit.

2. Such a structure has been visualized using single photon electron microscopy

Approach of the Study

1. Purified ATP synthase from Sacchromyces Cervesiae (yeast) and performed crystallization experiments.

2. Yeast ATP synthase after analysis with SDS page and HPLC found 13 subunits for ATP synthase: F1: α, β, γ, ε, δ

F0: a, b, c, d, h, f, OSCP, and ATP8

Methods of the Study

1. Crystals were prepared via the microbatch technique with polyethylene glycol as the precipitant.

2. Crystals were grown in the presence of ADP and a non-hydrolyzable ATP analog (AMP-PNP).

3. SDS page also showed that the subsequent crystals had only the α, β, γ, δ, ε, and c subunits but no other subunits (dissociation?)

4. The structure was solved with the stand alone version of AmoRe with from 15-5 Å and compared with the existing coordinates of the PDB file for the F1 subunit from bovine mitochondria.

1. The polar loops of c1-c5 subunits are covered by a δ subunit and two further associations between the γ stalk and consecutive subunits.

2. The point of contact may be due to side chain contacts between the main chain of the c subunits and β-barrel or the α-helices of the δ subunit

3. Electron densities of these c subunits are much stronger than un-associated c subunits.

1. The 10 c subunits form an almost symmetrical ring.

2. density for subunits 6, 7, and 8 is weaker, as seen from the asymmetrical appearance of the electron density

3. Increased density in the upper two thirds is probably due to phospholipids occupancy.

1. Atypical crystal packing for membrane proteins. The hydrophobic portion of the protein (c subunit) is bound to the solution exposed F1

2. The membrane region is associated with the “so called” F1

3. Likely an anomaly due to covering by detergents.

My thinking is that the association is being made between the some region of the δ subunit and the F1 as this does is possible according to the peripheral assembly model and single photon visualizations.

FunctionalFunctional Implications of the StructureStructure of F0

C ring stoichiometry

1. An unexpected finding was that there were 10 c subunits instead of 12 as previously anticipated

2. There may have been 12 and two were lost but that is unlikely because of the strong associations and the tight packing the c subunits form with eachother.

3. The atypical crystal packing would have selected for them anyway as they would pack much better than the 10 subunit species..I DISAGREE!!I DISAGREE!!

4. C ring stoichiometry is critical for understanding the amount of protons that need to be translocated to cause the 120° rotation of each γ subunit.

5. This means that the stoichiometry of the H+/ATP is non-integral abd is not 3 or 4 protons but somewhere in between. I DISAGREE!!

The electron density shown provides evidence that c protomers form a ring thatis in close contact with the γ and δ subunits.

Consistent with previousinterpretations, in which subunits γ, δ, and ε form a rotating ensemble.

It strongly supports the idea that the c oligomer is part of a rotarymotor, converting electrochemical energy into chemical energy stored in ATP.

To understand the mechanism of the generation of rotation (FUNCTION)(FUNCTION), it will be necessary to establish the nature of the interaction between the c subunit oligomer (Structure)(Structure) found here and the remaining subunits in F0.

Conclusion