Crystal Structures of Mutant Forms of the Yeast F1 ATPase Reveal Two Modes of Uncoupling

Arsenieva, Diana; Symerský, J.; Wang, Yamin; Pagadala, Vijayakanth; Mueller, David M.

doi:10.1074/jbc.m110.174383

Cited by 19 publications

(27 citation statements)

References 40 publications

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“…We found two significant sites in the ATP6 gene, which codes for subunit a , that is thought to participate directly in the proton flow [35]. Because of the difficulty in crystallizing membrane proteins, little information is known about the structure of the F0 proton channel [36] and therefore we have not conducted further structural analyses.…”

Section: Resultsmentioning

confidence: 99%

Evidence of Positive Selection in Mitochondrial Complexes I and V of the African Elephant

et al. 2014

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As species evolve, they become adapted to their local environments. Detecting the genetic signature of selection and connecting that to the phenotype of the organism, however, is challenging. Here we report using an integrative approach that combines DNA sequencing with structural biology analyses to assess the effect of selection on residues in the mitochondrial DNA of the two species of African elephants. We detected evidence of positive selection acting on residues in complexes I and V, and we used homology protein structure modeling to assess the effect of the biochemical properties of the selected residues on the enzyme structure. Given the role these enzymes play in oxidative phosphorylation, we propose that the selected residues may contribute to the metabolic adaptation of forest and savanna elephants to their unique habitats.

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Section: Resultsmentioning

confidence: 99%

Evidence of Positive Selection in Mitochondrial Complexes I and V of the African Elephant

et al. 2014

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“…S2). The latter two residues are part of a key region close to the β 394 DELSEED 400 loop, which has important interactions with the central γ-subunit (27)(28)(29). The transition to the second conformational state takes place when restraints are introduced after ∼16 ns.…”

Section: Resultsmentioning

confidence: 99%

Rate of hydrolysis in ATP synthase is fine-tuned by α-subunit motif controlling active site conformation

Beke‐Somfai

Lincoln

Nordén

2013

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

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Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F o F 1 ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F 1 performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.enzyme catalysis | molecular dynamics | protein structure M ost cellular processes depend on ATP as a free energy source. Among a wide spectrum of organisms, the key enzyme responsible for maintaining the ATP concentration is F o F 1 ATP synthase, with synthesis being driven by a transmembrane electrochemical gradient (1). The chemical reaction takes place in the F 1 domain at active sites located in the three αβ-subunit pairs surrounding a central α-helical coiled coil, the γ-subunit. It has been shown in single-molecule experiments on F 1 that ATP synthesis and ATP hydrolysis are coupled to γ-subunit rotations of opposite directions (1, 2). Early kinetic studies revealed a strong ATP concentration dependence for the hydrolysis rate that was explained involving both unisite and multisite catalysis: low ATP concentrations, with only one active site populated by unisite catalysis (3, 4), whereas at physiological (high) ATP concentrations, multisite catalysis takes over, resulting in several orders of magnitude increase in hydrolysis rate (5). This remarkable catalytic cooperativity of the enzyme could be an effect of the mechanochemical coupling between the active sites. In crystal structures, three conformations of the catalytically active sites can be seen, assigned as tight binding site (α TP /β TP ; T), loose binding site (α DP /β DP ; D), and empty site (α E / β E ; E) (6-10). During ATP hydrolysis, each α/β-subunit pair changes conformation along the cycle: E→T→D→E, each step associated with a 120°rotation of the γ-subunit (5, 7, 10, 11). Single-molecule studies supported by simulations indicate that the 120°rotation...

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“…By introducing the mgi mutations into the S. cerevisiae F 1 complex, the Mueller group found that the mutant ATP synthase is uncoupled (19). Four mutant F 1 complexes were crystallized, and their structures were analyzed (20). Interestingly, the mgi mutations were found either to affect substrate (phosphate) binding or to reduce the steric hindrance imposed by the ␤ subunit when ␥ rotates in the ␣ 3 ␤ 3 core.…”

Section: Remains Active In Hydrolyzing Atpmentioning

confidence: 99%

Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F ₁ -ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

Clark-Walker

Wang

et al. 2013

Eukaryot Cell

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F 1 -ATPase is a rotary molecular machine with a subunit stoichiometry of ␣ 3 ␤ 3 ␥ 1 ␦ 1 1 . It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central ␥ rotor dictates the sequential conformational changes to the catalytic sites in the ␣ 3 ␤ 3 core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F 1 -ATPase have suggested that the ␣ 3 ␤ 3 core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this ␥-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the ␣ and ␤ subunits that stimulate ATP hydrolysis by the mitochondrial F 1 -ATPase in the absence of ␥. We found that the F446I mutation in the ␣ subunit and G419D mutation in the ␤ subunit suppress cell death by the loss of mitochondrial DNA ( o ) in a Kluyveromyces lactis mutant lacking ␥. In organello ATPase assays showed that the mutant but not the wild-type ␥-less F 1 complexes retained 21.7 to 44.6% of the native F 1 -ATPase activity. The ␥-less F 1 subcomplex was assembled but was structurally and functionally labile in vitro. Phe446 in the ␣ subunit and Gly419 in the ␤ subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent ␣ or ␤ subunit, thereby allowing robust ATP hydrolysis and cell survival under o conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the ␣ 3 ␤ 3 subcomplex.

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Crystal Structures of Mutant Forms of the Yeast F1 ATPase Reveal Two Modes of Uncoupling

Cited by 19 publications

References 40 publications

Evidence of Positive Selection in Mitochondrial Complexes I and V of the African Elephant

Evidence of Positive Selection in Mitochondrial Complexes I and V of the African Elephant

Rate of hydrolysis in ATP synthase is fine-tuned by α-subunit motif controlling active site conformation

Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F ₁ -ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

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Crystal Structures of Mutant Forms of the Yeast F1 ATPase Reveal Two Modes of Uncoupling

Cited by 19 publications

References 40 publications

Evidence of Positive Selection in Mitochondrial Complexes I and V of the African Elephant

Evidence of Positive Selection in Mitochondrial Complexes I and V of the African Elephant

Rate of hydrolysis in ATP synthase is fine-tuned by α-subunit motif controlling active site conformation

Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F 1 -ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

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Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F ₁ -ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor