Cardiolipin binds selectively but transiently to conserved lysine residues in the rotor of metazoan ATP synthases

Abstract

The anionic lipid cardiolipin is an essential component of active ATP synthases. In metazoans, their rotors contain a ring of eight c-subunits consisting of inner and outer circles of N-and C-terminal α-helices, respectively. The beginning of the C-terminal α-helix contains a strictly conserved and fully trimethylated lysine residue in the lipid headgroup region of the membrane. Larger rings of known structure, from c 9 -c 15 in eubacteria and chloroplasts, conserve either a lysine or an arginine residue in the equivalent position. In computer simulations of hydrated membranes containing trimethylated or unmethylated bovine c 8 -rings and bacterial c 10 -or c 11 -rings, the head-groups of cardiolipin molecules became associated selectively with these modified and unmodified lysine residues and with adjacent polar amino acids and with a second conserved lysine on the opposite side of the membrane, whereas phosphatidyl lipids were attracted little to these sites. However, the residence times of cardiolipin molecules with the ring were brief and sufficient for the rotor to turn only a fraction of a degree in the active enzyme. With the demethylated c 8 -ring and with c 10 -and c 11 -rings, the density of bound cardiolipin molecules at this site increased, but residence times were not changed greatly. These highly specific but brief interactions with the rotating c-ring are consistent with functional roles for cardiolipin in stabilizing and lubricating the rotor, and, by interacting with the enzyme at the inlet and exit of the transmembrane proton channel, in participation in proton translocation through the membrane domain of the enzyme.ardiolipin is associated uniquely with energy-transducing membranes in mitochondria and eubacteria. In mitochondria, it is found in the inner membrane and is synthesized close to, or in, the inner leaflet (1) where most of it remains (2, 3). Cardiolipin consists of two 3-phosphatidyl groups linked by a glycerol bridge, and in bovine mitochondria, the four acyl chains have 18 carbon atoms with one or two unsaturated linkages (4). It has been proposed that under physiological conditions, the central hydroxyl and the two phosphates trap a proton in a resonance structure and that cardiolipin carries one net negative charge (5). However, re-evaluations of the pK a values of the phosphates indicate that under physiological conditions, the head-group of cardiolipin bears two negative charges (6, 7).Cardiolipin and other phospholipids are essential components of active ATP synthases isolated from mitochondria (8-12). It has been suggested that cardiolipin acts to stabilize and lubricate the rotating c-ring (13) or to aid in proton transfer (5), but it is not known where or how cardiolipin binds to the enzyme. Excluding the regulatory protein IF 1, the bovine ATP synthase complex is built from 28 polypeptide chains of 16 varieties (14). About 85% of a mosaic overall structure has been determined to atomic resolution by structural analysis of constituent domains (15-18), and an intact enzyme ...

Structure of the mitochondrial ATP synthase from Pichia angusta determined by electron cryo-microscopy

“…Also to be taken into consideration is the role of lipids in the mechanism of the enzyme. Cardiolipin bearing two negative charges is an essential component of active F-ATPases in bacteria and mitochondria (42). It appears to be attracted to the c-ring selectively over phospholipids, where it binds transiently and repeatedly around basic residues in the headgroup regions of both leaflets of the bilayer, suggesting a possible role in proton translocation (42).…”

“…Cardiolipin bearing two negative charges is an essential component of active F-ATPases in bacteria and mitochondria (42). It appears to be attracted to the c-ring selectively over phospholipids, where it binds transiently and repeatedly around basic residues in the headgroup regions of both leaflets of the bilayer, suggesting a possible role in proton translocation (42). As this paper neared completion, the structure of the dimeric F-ATPase from the fungus Yarrowia lipolytica determined by cryo-EM was published (43).…”

Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase

“…The N-terminal regions extend toward the space between the inner and outer membranes of the organelle, and each N-terminal region has aspartic acid residues at positions 1 and 3 and an unmodified lysine at position 7. Residues in the polar loops and the N-terminal extensions attract cardiolipin molecules selectively over phospholipids (44). With the exception of glutamate-58 on the outer surface of the ring, which plays a central role in the pathway for membrane translocation of protons, the outer surface of the ring is almost entirely hydrophobic, in keeping with its contact with the hydrophobic environment of the inner mitochondrial membrane.…”

Cardiolipin puts the seal on ATP synthase

“…In PNAS, Duncan et al (5) report that an unusual lipid-protein interaction in the F o part of ATP synthase is central to efficient c-ring rotation with properly sealed c-ring/a-subunit/membrane interfaces (Fig. 1A).…”

“…In CG-MD simulations using the MARTINI force field (5,17,18), groups of usually four heavy atoms are represented by a single interaction center. Such coarse graining describes lipidprotein interaction at a reasonable level of detail and makes it possible to simulate biomolecular systems on time scales normally inaccessible to fully atomistic simulations.…”