Does F₁‐ATPase subunit γ turn in the wrong direction?

Abstract

Analyzing the direction of F 1 -ATPase subunit Q Q rotation, its shape and non-random distribution of surface residues, a mechanism is proposed for how Q Q induces the closing/ opening of the catalytic sites at L L/K K interfaces: by keeping contact with the mobile domain of subunits L L at the 'jaw' (D386, the seven consecutive hydrophobic residues and D394/ E395), rotating Q Q works as a screw conveyer within the barrel of (K K,L L) 3 3 . This rotation, in turn, is energized by a rotation of the connected membrane integral rotor of F o c subunits driven by H þ e¥ux. A topological problem arises, however, when the information on (1) the direction of the rotation of subunits Q/O in the bacterial enzyme(s) during ATP hydrolysis [14,15] and (2) the X-ray structure of the bovine enzyme [16^18] is correlated with (3) the sequence of kinetic conformational states [2^9].In the X-ray structure of bovine F 1 , di¡erent sectors of the asymmetric subunit Q are in contact with the three subunits L in di¡erent conformations [16]. The relative position of the occupancies E (empty), TP (triphosphate binding) and DP (diphosphate binding) of the three L subunits is counter-clockwise, when viewed from above. Therefore, if the interactions with subunit Q determine the conformation and occupancy of the three di¡erent subunits L, the direction of rotation of subunit Q driven by ATP hydrolysis could have been deduced to be clockwise relative to the ¢xed catalytic sites, viewed from above. Accordingly, in videograms of ATP hydrolysisdriven movements of devices ¢xed to subunit Q the direction of rotation is clockwise, viewed from above, because it is counter-clockwise, when viewed from the membrane side [14,15,19].Mainly from exchange rates in pulse/chase experiments, on the other hand, three conformational states have been de¢ned, Open with low, Loose with medium and Tight with high a⁄nity to nucleotides, and the 'energy-linked binding change mechanism' has been formulated [2^7]. In the assignment [16] of the occupancies to these conformational states, E corresponds to O, TP to L and DP to T. This assignment suggests that the sequence of conformational changes during ATP hydrolysis is O-L-T-O. From kinetic analyses this O-L-T-O sequence has been deduced for ATP synthesis [2^10,20], however.The assumption that in the videograms Q turns arti¢cially in the wrong way would lead in reality to the sequence O-T-L-O in ATP hydrolysis, in agreement with the deduced sequence of the three kinetic conformational states; but this consideration would also require the interchange of the occupancies L DP and L TP in the X-ray structures. Interchanging the assignment [21] or introducing the additional conformation 'closed' into the static three-sector cartoons [10] does not solve the problem either. The 'direction of rotation discrepancy ' [22] is represented in this article as two three-step cartoons (Fig. 1a,b), showing the course of events in one catalytic site correlated to the three conformational a⁄nities. The data and conclusions rep...

Domain Motion of Individual F1-ATPase β-Subunits during Unbiased Molecular Dynamics Simulations

“…The closed β -subunit β TP unit was often assumed to have the highest ATP affinity 1 but later studies argued that the β DP site has the highest ATP affinity. 2 Recently it was found experimentally 3 that the β TP site is indeed the high-affinity site in agreement with the studies by Yang et al 4 …”

“…Nevertheless the high charge state of the individual β -subunits suggests that their conformation is influenced significantly by the electrostatic interaction with its environment, particularly with the γ -stalk and especially its jaw. 2 …”

“…Some of these studies investigated the structure-function relationship mainly based on structural information from X-ray studies and biochemical experiments. 2,5–8 Oster et al have performed studies based on structural interpolation, 9–11 some combined with molecular dynamics (MD) simulations. 12,13 In one of these studies 13 it was concluded that a β -sheet within the β -subunit acts as a spring that stores a considerable amount of elastic energy.…”

Making ATP

“…Conversely, in synthesis mode, the rotation of the ␥ shaft drives the ␤-subunits through their hinge-bending cycle. Several authors have found evidence that the mechanical coupling between the ␤ and ␥ subunits is tightened by a circumferential ''track'' of hydrophobic and positively charged residues around the ␥ shaft that guides the tips of the ␤-subunits (43,44). This track is shown in Fig.…”

“…To ensure the chemical transitions occur in the correct sequence, additional residue interactions must be included. By examining (43). Some other important residues are also shown: ␥R36 is the most eccentric residue, labeled MEP (i.e., furthest from the axis of rotation); ␥Q255 activates binding of nucleotide successively by interacting with the ␤-subunit; ␥R242 activates phosphate release in the hydrolysis direction by interacting with DELSEED; ␥M23 also interacts with DELSEED via chargehydrophobic interactions.…”