Structure of a bacterial ATP synthase

Abstract

ATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed the Bacillus PS3 ATP synthase in Eschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunit e shows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.structures of the membrane-embedded FO regions from mitochondrial and chloroplast ATP synthases to be determined to near-atomic resolutions [12][13][14][15] .Compared to their mitochondrial counterparts, bacterial ATP synthases have a simpler subunit composition. The F1 region consists of subunits a3b3gde, while the FO region is usually formed by three subunits with the stoichiometry ab2c9-15. Chloroplasts and a few bacteria, such as Paracoccus denitrificans, possess two different but homologous copies of subunit b, named subunits b and b¢ 6 . Each copy of subunit a and b contains a nucleotide binding site. The noncatalytic a subunits each bind to a magnesium ion (Mg 2+ ) and a nucleotide, while the catalytic b subunits can adopt different conformations and bind to Mg-ADP (bDP), Mg-ATP (bTP), or remain empty (bE). Crystal structures of bacterial F1-ATPases and c-rings from the FO regions of several species have been determined [16][17][18][19][20][21][22][23][24] . Structures of intact ATP synthases from E. coli have been determined to overall resolutions of 6 to 7 Å by cryo-EM, with the FO region showing lower quality than the rest of the maps, presumably due to conformational flexibility 25 . In structures of both intact ATP synthase 25 and dissociated F1-ATPase 17,19 from bacteria, subunit e adopts an "up" conformation that inhibits the ATP hydrolysis by the enzyme. In the thermophilic bacterium Bacillus PS3, this subunit e mediated inhibition is dependent on the concentration of free ATP 26-28 . Low ATP concentrations (eg. < 0.7 mM) promote the inhibitory up conformation while a permissive "down" conformation can be induced by a high concentration of ATP (eg. > 1 mM). This mechanism would allow the Bacillus PS3 ATP synthase to run in reverse, establishing a proton motive force by ATP hydrolysis, when the ATP concentration is sufficient to do so without depleting the cell's supply of ATP. In E. coli, however, in the absence of a sufficient proton motive force to drive ATP synthesis, inhibition of ATP hydrolysis by subunit e persists even when the concentration of ...

The mechanical inhibition of the isolated Vofrom V-ATPase for proton conductance

“…This salt bridge is conserved in both eukaryotic and prokaryotic V o (25,26). In contrast the salt bridge forms between a single arginine residue and a single glutamic (or aspartic) acid residue in F o ( 5, 30, 31 ). Similar to the two channel model described for other rotary ATPases ( 32, 33 ), the two arginine residues on the MH7 and 8 play an important role in protonation and deprotonation of the carboxy groups on the c 12 ring, with the resulting rotation of dc 12 driven by proton translocation from periplasmic to cytoplasmic sides.…”

Cryo-EM studies of the rotary H⁺-ATPase/synthase from <i>Thermus thermophilus</i>

“…For the V/A-ATPase, X-ray crystallography has been used to determine partial structures of the central axis [18,19], peripheral stalk [20], membrane-embedded ring of c-subunits [21], hydrophilic domain of a-subunit [22], A 3 B 3 hexamer [23], and soluble V 1 region [24]. Recent breakthroughs in structure analysis using cryogenic electron microscopy (cryo-EM) allowed us to determine the whole structure of rotary ATPases, including the F-type ATPase, V-ATPase, and V/A-ATPase [25][26][27][28][29][30][31][32][33]. In fact, the structure of the (0.34 nm) hexagonal lattice of carbon atoms with high electronic conductivity properties [41,42].…”

“…Recent breakthroughs in structural studies using cryo-EM by improvements in the detector and image processing methods allowed for determination of the whole structure of rotary ATPases [13,[27][28][29][30][31][32][33]. In 2015, the different rotational states of intact rotary ATPases were first observed in both the yeast V-ATPase and F-type ATP synthase, at 6-10 Å resolution, which has enabled a more detailed understanding of the molecular mechanism of these rotary motor proteins [28,29,33].…”

“…A nanodisc is a powerful tool for the removal of detergents which tend to decrease cryo-image contrast [32,49,50]. Additionally, series of new class of amphipol are available for cryo-grid preparation to obtain clearer cryo-micrographs [31,51,52]. Methodologies of grid preparation have also been improved by using a new material [53][54][55][56].…”

Characterization of regulatory central stalk epitopes of mycobacterial- and acetogen F-ATP synthases

“…Last but not least, my eternal gratitude goes to my late grandfather, Prof. Vidosav Marinković, whose persona and devotion inspired me and prepared for the challenges ahead. Tables Table 2. (1). In mycobacterial cells, ATP synthesis occurs by utilizing the proton-motive force (PMF) to drive the catalytic events in the F-ATP synthase and form ATP from ADP and P i , similarly to the mechanism found in other bacterial F-ATP synthases.…”