Structure of the F 1-binding Domain of the Stator of Bovine F 1F o-ATPase and How it Binds an α-Subunit (original) (raw)

How the N-terminal Domain of the OSCP Subunit of Bovine F 1F o-ATP Synthase Interacts with the N-terminal Region of an Alpha Subunit

Journal of Molecular Biology, 2007

The peripheral stalk of ATP synthase acts as a stator holding the α3β3 catalytic subcomplex and the membrane subunit a against the torque of the rotating central stalk and attached c ring. In bovine mitochondria, the N-terminal domain of the oligomycin sensitivity conferral protein (OSCP-NT; residues 1–120) anchors one end of the peripheral stalk to the N-terminal tails of one or more α subunits of the F1 subcomplex. Here, we present an NMR characterisation of the interaction between OSCP-NT and a peptide corresponding to residues 1–25 of the α-subunit of bovine F1-ATPase. The interaction site contains adjoining hydrophobic surfaces of helices 1 and 5 of OSCP-NT binding to hydrophobic side-chains of the α-peptide.

F1 and F0 connections in the bovine mitochondrial ATP synthase: The role of the of α subunit N-terminus, oligomycin-sensitivity conferring protein (OCSP) and subunit d

Eur J Biochem, 2001

We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F 1 F 0 complex and isolated F 1 of the bovine heart mitochondrial ATP synthase (EC 3.6.1.34). Chemical cross-linking of oligomycin-sensitivity conferring protein (OSCP) with other subunits of the ATP synthase and the consequent functional effects were also investigated. The results obtained show that the a subunit N-terminus is essential for the correct, functional connection of F 1 to F 0. The a-subunit N-terminus contacts OSCP which, in turn, contacts the F 0 I-PVP(b) and the F 0-d subunits. The N-terminus of subunit a, OSCP, a segment of subunit d and the C-terminal and central region of F 0 I-PVP(b) subunits are peripherally located with respect to subunits g and d which are completely shielded in the F 1 F 0 complex against trypsin digestion. This qualifies the N-terminus of subunit a, OSCP, subunit d and F 0 I-PVP(b) as components of the lateral element of the stalk. These subunits, rather than being confined at one side of the complex which would leave most of the central part of the g subunit uncovered, surround the g and the d subunits located in the central stalk.

F1 and F0 connections in the bovine mitochondrial ATP synthase: the role of the of alpha subunit N-terminus, oligomycin-sensitivity conferring protein (OCSP) and subunit d

European Journal of Biochemistry, 2000

We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F 1 F 0 complex and isolated F 1 of the bovine heart mitochondrial ATP synthase (EC 3.6.1.34). Chemical cross-linking of oligomycin-sensitivity conferring protein (OSCP) with other subunits of the ATP synthase and the consequent functional effects were also investigated. The results obtained show that the a subunit N-terminus is essential for the correct, functional connection of F 1 to F 0. The a-subunit N-terminus contacts OSCP which, in turn, contacts the F 0 I-PVP(b) and the F 0-d subunits. The N-terminus of subunit a, OSCP, a segment of subunit d and the C-terminal and central region of F 0 I-PVP(b) subunits are peripherally located with respect to subunits g and d which are completely shielded in the F 1 F 0 complex against trypsin digestion. This qualifies the N-terminus of subunit a, OSCP, subunit d and F 0 I-PVP(b) as components of the lateral element of the stalk. These subunits, rather than being confined at one side of the complex which would leave most of the central part of the g subunit uncovered, surround the g and the d subunits located in the central stalk.

F1 and F0 connections in the bovine mitochondrial ATP synthase

European Journal of Biochemistry, 2000

We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F 1 F 0 complex and isolated F 1 of the bovine heart mitochondrial ATP synthase (EC 3.6.1.34). Chemical cross-linking of oligomycin-sensitivity conferring protein (OSCP) with other subunits of the ATP synthase and the consequent functional effects were also investigated.

Stepwise Propagation of the ATP-induced Conformational Change of the F1-ATPase β Subunit Revealed by NMR

Journal of Biological Chemistry, 2008

The rotation of F 1-ATPase (F 1) is driven by the open/close bending motion of the ␤ subunit. The mechanism underlying the bending motion was investigated for the F 1 ␤ monomer from thermophilic Bacillus PS3 (TF 1 ␤) in solution, using mutagenesis and NMR. The hydrogen bond networks involving the side chains of Lys-164 (numbering for TF 1 ␤; 162 for mitochondrial F 1 ␤ in parentheses), Thr-165(163), Arg-191(189), Asp-252(256), Asp-311(315), and Arg-333(337) in the catalytic region are significantly different for the ligand-bound and free ␤ subunits in the crystal structures of mitochondrial F 1. The role of each amino acid residue was examined by Ala substitution. ␤(K164A) reduced the affinity constant for 5-adenyl-␤,␥-imidodiphosphate by 20-fold and abolished the conformational change associated with nucleotide binding and the ATPase activity of ␣ 3 ␤(K164A) 3 ␥. ␤(T165A) and ␤(D252A) exhibited no effect on the binding affinity but abolished the conformational change and the ATPase activity. The chemical shift perturbation of backbone amide signals of the segmentally labeled ␤(mutant)s indicated stepwise propagation of the open/close conversion on ligand binding. The key action in the conversion is the switching of the hydrogen-bonding partner of Asp-252 from Lys-164 to Thr-165. Residual dipolar coupling analysis revealed that the closed conformation of the ␤ monomer was more closed than that in the crystal structure and was different for MgATP-and MgADP-bound ␤ subunits. Actually, MgATP induced a conformational change around Tyr-307 (311 for MF 1 ␤), whereas MgADP did not. The significance of these findings is discussed in connection with the catalytic rotation of F 1-ATPase.

Ground state structure of F1-ATPase from bovine heart mitochondria at 1.9 Å resolution

Journal of Biological …, 2007

The structure of bovine F 1 -ATPase, crystallized in the presence of AMP-PNP and ADP, but in the absence of azide, has been determined at 1.9 Å resolution. This structure has been compared with the previously described structure of bovine F 1 -ATPase determined at 1.95 Å resolution with crystals grown under the same conditions but in the presence of azide. The two structures are extremely similar, but they differ in the nucleotides that are bound to the catalytic site in the ␤ DPsubunit. In the present structure, the nucleotide binding sites in the ␤ DP -and ␤ TP -subunits are both occupied by AMP-PNP, whereas in the earlier structure, the ␤ TP site was occupied by AMP-PNP and the ␤ DP site by ADP, where its binding is enhanced by a bound azide ion. Also, the conformation of the side chain of the catalytically important residue, ␣Arg-373 differs in the ␤ DP -and ␤ TP -subunits. Thus, the structure with bound azide represents the ADP inhibited state of the enzyme, and the new structure represents a ground state intermediate in the active catalytic cycle of ATP hydrolysis.

Conformational analysis of a mitochondrial presequence derived from the F1-ATPase β-subunit by CD and NMR spectroscopy

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1992

Previous studies on mitochondrial targeting presequences have indicated that formation of an amphiphillic helix may be required for efficient targeting of the precursor protein into mitochondria, but the structural details are not well understood. We have used CD and NMR spectroscopy to characterize in detail the structure of a synthetic peptide corresponding to the presequence for the /3-subunit of F~-ATPase, a mitochondrial matrix protein. Although this peptide is essentially unstructured in water, a-helix formation is induced when the peptide is placed in structure-promoting environments, such as SDS micelles or aqueous trifluoroethanol (TFE). In 50% TFE (by volume), the peptide is in dynamic equilibrium between random coil and a-helical conformations, with a significant population of a-helix throughout the entire peptide. The helix is somewhat more stable in the N-terminal part of the presequence (residues 4-10), and this result is consistent with the structure proposed previously for the presequence of another mitochondrial matrix protein, yeast cytochrome oxidase subunit IV. Addition of increasing amounts of TFE causes the a-helical content to increase even further, and the TFE titration data for the presequence peptide of the F~-ATPase/3-subunit are not consistent with a single, cooperative transition from random coil to a-helix. There is evidence that helix formation is initiated in two different regions of the peptide. This result helps to explain the redundancy of the targeting information contained in the presequence for the F~-ATPase/3-subunit.

The ?? complexes of ATP synthase: the ?3?3 oligomer and ?1?1 protomer

Journal of Bioenergetics and Biomembranes, 1992

The basic structures of the catalytic portion (F1, e313376e) of ATP synthase are the ~3 f13 hexamer (oligomer with cooperativity) and c¢1fll heterodimer (protomer). These were reconstituted from the c~ and fl subunits of thermophilic F~ (TF1), and the 0~3]~ 3 hexamer was crystallized. On electrophoresis, both the dimer and hexamer showed bands with ATPase activity. Using the dimer and hexamer, we studied the nucleotide-dependent rapid molecular dynamics. The formation of the hexamer required neither nucleotide nor Mg. The hexamer was dissociated into the dimer in the presence of MgADP, while the dimer was associated into the hexamer in the presence of MgATP. The hexamer, like mitochondrial F~ and TF~, showed two kinds of ATPase activity: one was cooperative and was inhibited by only one BzADP per hexamer, and the other was inhibited by three BzADP per hexamer.