Insights into the molecular mechanism of rotation in the Fo sector of ATP synthase - PubMed (original) (raw)

Insights into the molecular mechanism of rotation in the Fo sector of ATP synthase

Aleksij Aksimentiev et al. Biophys J. 2004 Mar.

Abstract

F(1)F(o)-ATP synthase is a ubiquitous membrane protein complex that efficiently converts a cell's transmembrane proton gradient into chemical energy stored as ATP. The protein is made of two molecular motors, F(o) and F(1), which are coupled by a central stalk. The membrane unit, F(o), converts the transmembrane electrochemical potential into mechanical rotation of a rotor in F(o) and the physically connected central stalk. Based on available data of individual components, we have built an all-atom model of F(o) and investigated through molecular dynamics simulations and mathematical modeling the mechanism of torque generation in F(o). The mechanism that emerged generates the torque at the interface of the a- and c-subunits of F(o) through side groups aSer-206, aArg-210, and aAsn-214 of the a-subunit and side groups cAsp-61 of the c-subunits. The mechanism couples protonation/deprotonation of two cAsp-61 side groups, juxtaposed to the a-subunit at any moment in time, to rotations of individual c-subunit helices as well as rotation of the entire c-subunit. The aArg-210 side group orients the cAsp-61 side groups and, thereby, establishes proton transfer via aSer-206 and aAsn-214 to proton half-channels, while preventing direct proton transfer between the half-channels. A mathematical model proves the feasibility of torque generation by the stated mechanism against loads typical during ATP synthesis; the essential model characteristics, e.g., helix and subunit rotation and associated friction constants, have been tested and furnished by steered molecular dynamics simulations.

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Figures

FIGURE 1

Schematic view of the E. coli ATP synthase. The solvent-exposed F1 unit (top) consists of subunits α_3_β_3_γδɛ; the membrane Fo unit (bottom) consists of subunits _ab_2_c_10.

FIGURE 2

Microscopic model of Fo ATPase composed of a four-helix bundle (_a_2–_a_5) of subunit a and an oligomer of 10 _c_-subunits. Only backbones of subunit a and of one out of 10 _c_TMH-2 (_c_2R) are shown as tubes; the rest of the _c_10 oligomer's helices are shown as cylinders. The binding sites (_c_Asp-61) of the _c_10 oligomer, the termini of the proton half-channels (_a_Ser-204 and _a_Asn-214), and the critical _a_Arg-210 residues of subunit a are drawn in van der Waals representation. The interface between _a_- and _c_-subunits was modeled.

FIGURE 3

RMSD values of the _a_1_c_10 complex _α_-carbon atoms during the equilibration.

FIGURE 4

Stochastic model for Fo (view from cytoplasm). Four out of 10 _c_-subunits and the _a_-subunit are shown. The _c_10 complex is fixed, and the _a_-subunit can move in either direction (angle _θ_a). This is equivalent to the more natural choice of a fixed subunit a and a moving _c_10 complex. The second transmembrane helix (_c_2) of each _c_-subunit can rotate independently (described by angles _θ_1, _θ_2, _θ_3, and _θ_4), thereby moving the key _c_Asp-61 residues, which are the proton-binding sites. The _c_1 helices do not rotate. Similarly, only the fourth helix of the _a_-subunit (_a_4) can rotate (angle _θ_R), moving the _a_Arg-210 residue; helices _a_2, _a_3, and _a_5 do not rotate. Proton transfer occurs between the terminal residue of the periplasmic channel (_a_Asn-214) and the _c_Asp-61 binding site on helix _c_2R, and between the terminal residue of the cytoplasmic channel (_a_Ser-206) and the _c_Asp-61 binding site on _c_2L. Motions are confined to the plane of the figure. The system is fully described by helix orientations _θ_1, _θ_2, _θ_3, and _θ_4 (_c_-subunits), _θ_R (_a_4), rotor angle _θ_a, and protonation state of the two aspartates (_c_Asp-61) on helices _c_2L and _c_2R.

FIGURE 5

Potentials of mean force used in the stochastic simulations: a double-well potential governing rotation of the _c_2 helices (open squares) and a parabolic potential governing rotation of the _a_4 helix (open circles).

FIGURE 6

Schematic representation of the sequence of events suggested by our study. These events, labeled a_–_f, occur during rotation of the c_10 oligomer by 2_π/10 in the synthesis direction, viewed here from the cytoplasm. (a) In the starting conformation, two residues _c_Asp-61 are deprotonated and form a bidentate salt bridge with _a_Arg-210, _c_Asp-61−–_a_Arg-210–_c_Asp-61−. (b) A proton is transferred from the terminal residue of the periplasmic proton channel, _a_Asn-214, to _c_Asp-61 on helix _c_2R. (c) Subunit a rotates clockwise with respect to the _c_10 oligomer in concert with a clockwise rotation of helix _c_2L. When subunit a approaches helix _c_2L′, _c_Asp-61 on that helix rotates by 180°. The latter rotation may proceed in either clockwise or counterclockwise direction. (d) The concerted rotation of subunit a and helix _c_2L are completed: _c_Asp-61 on helix _c_2L′ has rotated by 180° toward subunit a. (e) A proton is transferred to the terminal residue of the cytoplasmic proton channel, _a_Ser-206. (f) The system returns to the starting conformation a, but with the c_10 oligomer advanced by an angle 2_π/10. We note that the processes depicted are of stochastic nature, and, hence, do not necessarily obey the strict sequence shown.

FIGURE 7

Hydrogen-bond network formed between the binding sites (_c_Asp-61) and the terminal residues of the proton periplasm (_a_Asn-214) and cytoplasm (_a_Ser-206) channels. The critical residue _a_Arg-210 forms transient hydrogen bonds with both binding sites.

FIGURE 8

Concerted rotation of the _c_-subunit outer helix and the _c_10 complex in a lipid bilayer. The _c_2L helix has been forced to rotate clockwise by 180°. Shown in the instance when the salt bridge is transferred between two neighboring _c_-subunits, i.e., when the conformation _c_Asp-61−–_a_Arg-210–_c_Asp-61− has been momentarily assumed.

FIGURE 9

Stochastic events involved in Fo function. (a) Time evolution of helix angles _θ_2 (black), _θ_3 (red), _θ_R (green), and rotor angle _θ_a (blue). The angles are defined in Fig. 4. The _a_-subunit rotation takes place in discrete steps (blue line). (b) Distances between residues _a_Arg-210–_c_Asp-61 of _c_2L (black) and _a_Arg-210–_c_Asp-61 of _c_2R (red). Respective salt bridges are formed when these distances decrease below ∼0.25 nm. When both aspartates are deprotonated, a two-color pattern of lines at 0.25 nm indicates a frequent transfer of the salt bridge from one aspartate to another. When one of the aspartates is protonated (highlighted regions), a two-color pattern does not indicate a salt bridge transfer, but originates from the cyclic boundary conditions invoked when subunit a passes the boundary. (c) Protonation states of _c_Asp-61L (black) and _c_Asp-61R (red) (see text). (d) Nonbonded interaction energy of the three residues. The steps of the energy function are correlated with protonation/deprotonation of two _c_Asp-61 residues and the step motion of the _a_-subunit.

FIGURE 10

Substeps of _a_-subunit rotation. (Left) When both _c_Asp-61 residues are deprotonated, the average internal potential acting on the _a_-subunit is symmetric (dashed line). The F1 load (dotted line) shifts the minimum of the average potential to the right (solid line). This figure corresponds to steps a and f in Fig. 6. (Right) When _c_Asp-61 on _c_2R receives a proton from the periplasm, the average internal potential becomes asymmetric. The minimum of the total potential is shifted to the left in this case. This figure corresponds to steps c and d in Fig. 6.

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Insights into the molecular mechanism of rotation in the Fo sector of ATP synthase - PubMed (original) (raw)