Ligand-dependent structural variations in Escherichia coli F1 ATPase revealed by cryoelectron microscopy.

Abstract

The Escherichia coli F, ATPase, ECFI, has been examined by cryoelectron microscopy after reaction with Fab' fragments generated from monoclonal antibodies to the a and E subunits. The enzyme-antibody complexes appeared triangular due to the superposition of three anti-a Fab' fragments on alternating densities of the hexagonally arranged a and .3 subunits. The Fab' to the E subunit superimposed on a .8 subunit. A density was observed near the center of the structure in the internal cavity. Mg2+ were added and ATP hydrolysis was allowed to proceed, almost two-thirds of the images were in the class in which the central density was closest to the 13 subunit superimposed by the E subunit. We conclude that domains within the ECF, structure, either the central mass or a domain including the E subunit, move in the enzyme in response to ligand binding. We suggest that this movement is involved in coupling catalytic sites to the proton channel in the FO part of the ATP synthase.Energy-transducing membranes, including the plasma membrane of bacteria, the thylakoid membrane of chloroplasts, and the mitochondrial inner membrane, all contain a structurally homologous F1Fo ATP synthase (reviewed in refs. 1 and 2). This enzyme complex uses the energy of the transmembrane proton gradient, generated by oxidative or photophosphorylation, to synthesize ATP from ADP and inorganic phosphate (Pi) (3,4).The ATP synthase is organized into two distinct subassemblies, a catalytically active portion extrinsic to the membrane (F1) and a bilayer-spanning proton channel (FO). The compositionally simplest ATP synthases are found in bacteria: the Escherichia coli F1 (ECF1) is made up of five subunits, a, /3, y, 8, and E, in the ratio 3:3:1:1:1, and the ECFo is composed of three subunits, a, b, and c, in a stoichiometry of 1:2:10-12 (4-7).The catalytic sites in F1 are located on the ,8 subunits (1, 2); hence there are potentially three active sites per F1. Catalysis is highly cooperative, in that release of products is very slow when only one site is occupied, and increases dramatically (up to 106-fold) by binding substrate at a second catalytic site (8)(9)(10). Cooperativity between active sites is likewise critical for ATP synthesis by F1FO (10,11).To explain both the high degree of cooperativity and the unusual stoichiometry of the enzyme, catalytic mechanisms involving alternating active sites have been proposed (12-14); the models differ in the number of active sites, their order of use, and the involvement of regulatory sites. In the intact ATP synthase, both ATP synthesis and hydrolysis are tightly coupled to proton flow through Fo (15). As Fo appears to contain only one proton channel, consisting in part of the single copy of the a subunit (1, 16-18), only one catalytic site can be coupled and active at one time. Hence any multiplesite mechanism requires a shift in the coupling of Fo to each of the catalytic sites in turn. Such a sequential linking of the proton channel to active sites has been suggested to involve rotation of pa...

Molecular imaging of Escherichia coli F0F1‐ATPase in reconstituted membranes using atomic force microscopy

“…2A: regardless of lae finite dimensions of the scanning tip, the measured height ,~ 100 ,~) of FoF1 complexes on AFM should reflect the real height of the sample assuming that flattening of the sample is l~finimal. The values are in good agreement with those esti1hated on electron microscopy [4][5][6]. However, the apparent t:orizontal dimensions of well isolated molecules determined by AFM are generally much larger than the actual dimensions lgr the following reasons: (i) The force exerted by the AFM tp is large enough to flatten the sample [15].…”

“…Sufficient structural information on the F1 sector has been obtained by electron microscopy [4][5][6] and X-ray crystallography [7,8]. The higherordered structure of the catalytic subunit complex is consistent with the binding-change mechanism of ATP synthesis [9].…”

Membrane Topology of Subunit a of the F1F0 ATP Synthase as Determined by Labeling of Unique Cysteine Residues

“…The crystallization of F 1 from bovine mitochondria (7) led to a high resolution structure of the ␣ 3 ␤ 3 hexamer, plus parts of ␥ in the central core. Electron cryomicroscopic images have also contributed to an understanding of subunit arrangement (8,9) and motion in F 1 subunits (10). Subsequently, the hypothesis of rotation of ␥ relative to ␣ 3 ␤ 3 (11) has been supported by studies involving engineered disulfide-cross-linking of ␤ to ␥ (12), a fluorescence technique termed "polarized absorption recovery after photobleaching" (13), and direct visualization of rotation of fluorescently labeled actin filaments covalently attached to ␥ (14).…”

A cryoelectron microscopy study of the interaction of the Escherichia coli F1‐ATPase with subunit b dimer

“…The fact that the b subunit does not extend into the cavity within the F, part leaves sufficient space for the extensive movements of the y and E subunits between catalytic sites that have been observed in cryoelectron microscopy studies [9,18].…”