Molecular Architecture of the Rotary Motor in ATP Synthase: Daniela Stock, Andrew G. W. Leslie, John E. Walker By: Shayna Patel

Background Information ATP synthase is the key component in oxidative phosphorylation through the process of cellular respiration or photosynthesis Responsible for ATP (energy) production in a cell Utilizes protons from an electrochemical gradient to initiate the creation of ATP ATP synthase is composed of two regions: F o and F 1 F 0 portion is embedded in the inner membrane F 1 portions is projected in the matrix (NOT in the membrane)

Basic Structure of ATP Synthase F 1 portion is linked to F o by a central stalk (y) 3 common subunits: a, b, c F 0 has a rotation generator that produces ATP Subunits of F 1 : α3 β3 γ1 δ1 ε1 Alpha and beta subunits alternate around a coil of 2 antiparallel alpha helices around gamma Catalytic portion: Beta and alpha/beta interface Little is known about F o structure Remainder of the y subunit protrudes from the alpha/beta3 subunits and can crosslink to polar loop region of the c subunits in F o

Abstract Summary ATP synthase has a rotary motor to allow the production of ATP in the F o portion which is fueled by the proton motive force The electron density map of yeast mitochondrial ATP synthase shows a ring of 10 c subunits (each c subunit has an alpha helical hairpin) Each interhelical loop of 6 to 7 c subunits are in close contact with the gamma (γ) and δ portion of the stalk, and this contact between the c ring and the stalk proposes that they rotate as an ensemble during catalysis When the central stalk rotates, conformational change occurs in three states of the Beta subunits; these three states correspond to either the release of product ATP, binding of ADP and P i or the formation of ATP (cycle is known as “binding change mechanism”)

Abstract Summary Production of each ATP requires 120 degree rotation of the y subunit; there is a link between the number of protons needed per ATP synthesized Can be achieved with 9 c subunits in the ring and 3 translocated protons OR 12 c subunits in the ring and 4 translocated protons A NMR structure of the monomer of the E.coli c subunit shows that the protein is folded into two alpha helices linked by a loop The COOH alpha helix contains a conserved side chain essential for proton translocation This rotary mechanism requires a peripheral stator to counter the tendency of alpha/beta 3 to follow the rotation of y The beta subunits of F o may form part of the stator

Proposed ATP Synthase Model

How the Structure was Determined Purified ATP synthase from S. cerecisiae mitochondria, then carried out crystallization experiments By using a SDS-PAGE, HPLC and NH2 terminal sequencing it was determined that the purified complex consisted of these subunits: OSCP, d, a, b, h, f, ATP8, c, alpha, beta, gamma, delta, epsilon Then when the crystals were grown in the presence of ADP and nonhydrolyzable ATP (AMP) the subunits detected were: alpha, beta, gamma, delta, epsilon and c by the SDS-PAGE ONLY! The other subunits dissociated from the complex during crystallization leaving subcomplex consisting of F 1 and 10 copies of subunit c F1 complex solved by viewing program AMoRe (TABLE)

Model Statistics

How the Structure was Determined The alpha helices of the c subunits and extensive additional density in the central stalk could be seen in the electron density map (next slide) Main crystal contacts are between the bottom of a ring of 10 c subunits and the pseudo-3-fold top of an adjacent F 1 assembly Hydrophobic external regions of the c oligomers (which are normally in contact with phospolipid) are not involved in any crystal contacts and are probably covered by unresolved molecules of detergent

Solvent Density Modification

Electron Density map of gamma, delta, c subunit (E.coli model)

Electron Density Map of c Subunit Ring

Crystal Packing of F1-c Complex

Rotation of ATP Synthase C1 to c5 are covered by the delta subunit Two loops (c2 and c3) from the c subunit are in intimate contact involving hydrogen bonds between pairs of main chain atoms; another three (c1, c4, c5) could form side chain contacts to either the Beta barrel of the delta subunit or the lower portion of its two alpha helices The gamma subunit can make side chain contacts to c9, c10 and c1 that is also in contact with the Beta barrel of the delta subunit Therefore, 6 or 7 consecutive subunits (about 2/3) of the top surface of the ring are in contact with the foot of the stalk Supports the hypothesis that the complex and c subunits rotates as an ensemble during catalysis

Principle of Symmetry Mismatch It is likely that the crystal contact between the ring of the c subunit and the pseudo-3- fold F1 domain would have selected for 12 c subunits, but since 10 c subunits matches perfectly it can be suggested that either some of the c subunits were lost during crystallization or were never there to begin with For the suggested 12 c subunit, the three fold symmetry of the F1 sector and the 10 fold symmetry of the c ring within one complex do not match up Example of symmetry mismatch is also found in some ATP dependent proteases, where a rotational mechanism has been suggested to contribute to protein unfolding Another example of symmetry mismatch is the DNA machinery of bacteriophages DNA is injected through the 6 fold symmetry tail into its head (the head-tail connector is believed to rotate with the head during DNA packaging) The symmetry mismatch between the head and the connector is thought to facilitate rotation by avoiding deeper energy minima that would accompany matching symmetries ( ATP synthase made 2 distinct regions: F0 and F1, AMAZING! )

Effect of Different Number of c Subunits The number of c subunits in the ring has profound implications for the number of protons translocated by F0 for each ATP molecule synthesized in F1 A ring with 10 c subunits in it suggests that the H/ATP ratio is probably nontintegral and that its value lies between 3 and 4 If you assume that the c ring and the gamma subunit rotate as an ensemble, a nonintegral H/ATP ratio also makes it more likely that there is some degree of elasticity in the gamma subunit The stepping of the c ring can be matched to the stepping in F1 It has been suggested that the number of c subunits in E.coli varies depending on the carbon source; more c subunits appear to assemble into the complex if cells are grown in glucose with succinate C subunit ring is smaller and this reduces the gearing ratio, which would change the membrane potential required for ATP synthesis

Results Electron density shown gives proof that c protomers form a ring that are in close contact with the gamma and delta subunits Strongly supports the idea that the c oligomer is part of a rotary motor, converting electrochemical energy into energy stored in ATP A c stoichiometry of 9 or 12 has been considered to require translocation of 3 or 4 protons per ATP synthesized to impart a 120 degree rotation to the c ring 2 regions of interaction 1. F1 involved the gamma subunit and the alpha/beta3 subcomplex 2. F 0 involves the ring of c subunits and the alpha subunit

ATP Synthase Video yM&feature=related yM&feature=related