How the regulatory protein, IF
            <sub>1</sub>
            , inhibits F
            <sub>1</sub>
            -ATPase from bovine mitochondria

Gledhill, Jonathan; Montgomery, M.G.; Leslie, Andrew G. W.; Walker, John E.

doi:10.1073/pnas.0707326104

Cited by 181 publications

(251 citation statements)

References 30 publications

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“…S3B). Significant differences in the structures and positions of the central stalk in various structures of F 1 -ATPase have been noted previously (3,5,6,18). Attempts have been made to interpret these positions in terms of the catalytic cycle of the enzyme, but it has been noted that the positions could be influenced by contacts between adjacent complexes in the lattice of the crystals used in the structure determination.…”

Section: Resultsmentioning

confidence: 98%

“…However, no phosphate is bound in this location in the other two copies of the enzyme in the asymmetric unit. In many ground-state structures of bovine F 1 -ATPase, density for phosphate (or its analog sulfate) has been observed in a different position to where it has been observed in the yeast enzyme (18), close to the P-loop region. However, when this electron density is modeled as a phosphate, the temperature factors are very high, and there are only a few interactions with the protein.…”

Section: Resultsmentioning

confidence: 99%

“…In Upper, the conformations of the β E -, β TP -and β DP -subunits are taken from the ground-state (GS) structure of bovine F 1 -ATPase. They are placed in the order described before (4,18), each conversion requiring a 120°r otary step of the central stalk of the enzyme. In the conversion of β E -GS to β TP -GS, the substrate, magnesium.ATP, is bound to the catalytic site.…”

Section: Methodsmentioning

confidence: 99%

“…However, many of the inhibitors arrest the catalytic cycle in the ground state (15)(16)(17), except that the natural inhibitor protein IF 1 arrests bovine F 1 -ATPase in either a posthydrolysis preproduct-release state, or, in a "dead-end" state of the enzyme (18). Another structure of bovine F 1 -ATPase inhibited by magnesium.ADP and AlF 4 − , designated F 1 -TS, represents a "transition state" intermediate in ATP hydrolysis (5), with magnesium.ADP-fluoroaluminate bound to both β DP -and β TPsubunits, and magnesium.ADP and sulfate (or phosphate) to the β E -subunit.…”

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confidence: 99%

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Structural evidence of a new catalytic intermediate in the pathway of ATP hydrolysis by F ₁ –ATPase from bovine heart mitochondria

Rees

Montgomery

Leslie

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The molecular description of the mechanism of F 1 -ATPase is based mainly on high-resolution structures of the enzyme from mitochondria, coupled with direct observations of rotation in bacterial enzymes. During hydrolysis of ATP, the rotor turns counterclockwise (as viewed from the membrane domain of the intact enzyme) in 120°steps. Because the rotor is asymmetric, at any moment the three catalytic sites are at different points in the catalytic cycle. In a "ground-state" structure of the bovine enzyme, one site (β E ) is devoid of nucleotide and represents a state that has released the products of ATP hydrolysis. A second site (β TP ) has bound the substrate, magnesium. ATP, in a precatalytic state, and in the third site (β DP ), the substrate is about to undergo hydrolysis. Three successive 120°turns of the rotor interconvert the sites through these three states, hydrolyzing three ATP molecules, releasing the products and leaving the enzyme with two bound nucleotides. A transition-state analog structure, F 1 -TS, displays intermediate states between those observed in the ground state. For example, in the β DP -site of F 1 -TS, the terminal phosphate of an ATP molecule is undergoing in-line nucleophilic attack by a water molecule. As described here, we have captured another intermediate in the catalytic cycle, which helps to define the order of substrate release. In this structure, the β E -site is occupied by the product ADP, but without a magnesium ion or phosphate, providing evidence that the nucleotide is the last of the products of ATP hydrolysis to be released.catalytic mechanism | magnesium release | nucleotide release H igh-resolution structures of F 1 -ATPase from bovine heart and yeast mitochondria have provided the molecular basis for the catalytic mechanism of ATP synthesis and hydrolysis by F 1 F oATPase (1-10). During ATP synthesis, a mechanical rotation of the γ-subunit driven by the transmembrane proton-motive force, exerted on a ring of C-subunits in the F o membrane domain of the intact enzyme, takes each of the three β-subunits in its catalytic domain through the states represented by the β TP -, β DP -, and β Esubunits, thereby synthesizing three ATP molecules during each 360°rotation (1, 11). Hydrolysis of ATP reverses the direction of rotation, and protons are ejected from the mitochondrial matrix into the intermembrane space through the F o domain of the enzyme (12, 13). The rotation of the γ-subunit is not continuous, but it proceeds in 120°steps consisting of 90°and 30°substeps (14). The ground-state structure of F 1 -ATPase (denoted F 1 -GS) probably represents the state of the enzyme at the end of the complete 120°rotary step.Attempts have been made to access structures that represent conformations of β-subunits that are intermediate between the three conformations in the ground-state structure by inhibiting the enzyme in various ways. However, many of the inhibitors arrest the catalytic cycle in the ground state (15-17), except that the natural inhibitor protein IF 1 arrests bovine F ...

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Structural evidence of a new catalytic intermediate in the pathway of ATP hydrolysis by F ₁ –ATPase from bovine heart mitochondria

Rees

Montgomery

Leslie

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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“…ATP synthase was isolated using metal affinity chromatography after binding to heterologously expressed residues 1-60 of the naturally occurring inhibitor protein (IF 1 ) with a C-terminal tag of six histidine residues (42). In the final step of the purification, the complex was eluted from the chromatography matrix in buffer containing 20 mM Tris·HCl (pH 7.8), 100 mM NaCl, 2 mM MgSO 4 , 1 mM ATP, 25 mM imidazole, 0.02% (wt/vol) NaN 3 , 10% (vol/vol) glycerol, 0.1% (wt/vol) DDM, and 0.1 mg/mL cardiolipin, phosphotidylethanolamine, and phosphotidylcholine (3:1:1) from bovine heart (Avanti Polar Lipids).…”

Section: Methodsmentioning

confidence: 99%

Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM

Baker

Watt

Runswick

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Mitochondrial ATP synthase is responsible for the synthesis of ATP, a universal energy currency in cells. Whereas X-ray crystallography has revealed the structure of the soluble region of the complex and the membrane-intrinsic c-subunits, little is known about the structure of the six other proteins (a, b, f, A6L, e, and g) that comprise the membrane-bound region of the complex in animal mitochondria. Here, we present the structure of intact bovine mitochondrial ATP synthase at ∼18 Å resolution by electron cryomicroscopy of single particles in amorphous ice. The map reveals that the a-subunit and c 8 -ring of the complex interact with a small contact area and that the b-subunit spans the membrane without contacting the c 8 -ring. The e-and g-subunits extend from the a-subunit density distal to the c 8 -ring. The map was calculated from images of a preparation of the enzyme solubilized with the detergent dodecyl maltoside, which is visible in electron cryomicroscopy maps. The structure shows that the micelle surrounding the complex is curved. The observed bend in the micelle of the detergent-solubilized complex is consistent with previous electron tomography experiments and suggests that monomers of ATP synthase are sufficient to produce curvature in lipid bilayers.A TP synthases are responsible for the synthesis of ATP from ADP and inorganic phosphate. In mammalian mitochondria, the enzyme is an ∼600-kDa membrane protein complex comprised of a catalytic F 1 region and a membrane-bound F O region. The F 1 region consists of subunits α 3 β 3 γδε (1, 2) and the F O region consists of subunits abc 8 defg(A6L)F 6 (3). The F 1 and F O regions are connected by a central stalk, comprised of the γ-, δ-, and ε-subunits from the F 1 region, and a peripheral stalk, comprised of the oligomycin sensitivity conferral protein (OSCP) and subunits b, d, and F 6 from the F O region (4, 5). Previously, electron cryomicroscopy (cryo-EM) of intact detergent-solubilized bovine ATP synthase particles embedded in a thin layer of amorphous ice revealed the overall shape of the complex at 32 Å resolution (6), and subsequent analysis of the Saccharomyces cerevisiae enzyme produced a similar map at 24 Å resolution (7). X-ray crystallography of subcomplexes of the bovine enzyme has defined the arrangement of subunits in the F 1 region (8) and the peripheral stalk (9, 10) and showed the presence of a ring of eight c-subunits in the F O region (11). The structure and arrangement of the remaining subunits in the membrane-bound region of the enzyme are not known.The ATP synthase functions by a rotary catalytic mechanism. Proton translocation through the F O region requires the a-, b-, and c-subunits (12-14) and induces rotation of the membranebound c 8 -ring (15). The structure of the c 8 -ring is thought to be stabilized by binding of cardiolipin to a lysine residue conserved throughout animalia that has been shown to be trimethylated at the ε-amino group in all animal ATP synthases tested (11,16,17). The c 8 -ring is attached to the central st...

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Ion Pumps and Molecular Motors: P‐, F‐, and V‐type ATPases

2009

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How the regulatory protein, IF ₁ , inhibits F ₁ -ATPase from bovine mitochondria

Cited by 181 publications

References 30 publications

Structural evidence of a new catalytic intermediate in the pathway of ATP hydrolysis by F ₁ –ATPase from bovine heart mitochondria

Structural evidence of a new catalytic intermediate in the pathway of ATP hydrolysis by F ₁ –ATPase from bovine heart mitochondria

Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM

Ion Pumps and Molecular Motors: P‐, F‐, and V‐type ATPases

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How the regulatory protein, IF 1 , inhibits F 1 -ATPase from bovine mitochondria

Cited by 181 publications

References 30 publications

Structural evidence of a new catalytic intermediate in the pathway of ATP hydrolysis by F 1 –ATPase from bovine heart mitochondria

Structural evidence of a new catalytic intermediate in the pathway of ATP hydrolysis by F 1 –ATPase from bovine heart mitochondria

Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM

Ion Pumps and Molecular Motors: P‐, F‐, and V‐type ATPases

Contact Info

Product

Resources

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How the regulatory protein, IF ₁ , inhibits F ₁ -ATPase from bovine mitochondria

Structural evidence of a new catalytic intermediate in the pathway of ATP hydrolysis by F ₁ –ATPase from bovine heart mitochondria

Structural evidence of a new catalytic intermediate in the pathway of ATP hydrolysis by F ₁ –ATPase from bovine heart mitochondria