Structure of a catalytic dimer of the α- and β-subunits of the F-ATPase fromParacoccus denitrificansat 2.3 Å resolution

Abstract

The structures of F-ATPases have predominantly been determined from mitochondrial enzymes, and those of the enzymes in eubacteria have been less studied.Paracoccus denitrificansis a member of the α-proteobacteria and is related to the extinct protomitochondrion that became engulfed by the ancestor of eukaryotic cells. TheP. denitrificansF-ATPase is an example of a eubacterial F-ATPase that can carry out ATP synthesis only, whereas many others can catalyse both the synthesis and the hydrolysis of ATP. Inhibition of the ATP hydrolytic activity of theP. denitrificansF-ATPase involves the ζ inhibitor protein, an α-helical protein that binds to the catalytic F1domain of the enzyme. This domain is a complex of three α-subunits and three β-subunits, and one copy of each of the γ-, δ- and ∊-subunits. Attempts to crystallize the F1–ζ inhibitor complex yielded crystals of a subcomplex of the catalytic domain containing the α- and β-subunits only. Its structure was determined to 2.3 Å resolution and consists of a heterodimer of one α-subunit and one β-subunit. It has no bound nucleotides, and it corresponds to the `open' or `empty' catalytic interface found in other F-ATPases. The main significance of this structure is that it aids in the determination of the structure of the intact membrane-bound F-ATPase, which has been crystallized.

Evolution of the Inhibitory and Non-Inhibitory ε, ζ, and IF1 Subunits of the F1FO-ATPase as Related to the Endosymbiotic Origin of Mitochondria

“…This confirms the partial and insufficient inhibitory effect of MgADP and the total and, therefore, enough inhibition exerted by ζ. The inhibitory mechanism of this subunit has been described in detail by our group and coworkers, firstly discovering the ζ subunit and describing its intrinsically disordered inhibitory N-terminal domain, subsequently demonstrating that ζ and IF 1 share the same inhibitory binding site at the α DP /β DP /γ interface of the F 1 -ATPase [ 14 , 29 , 30 , 41 , 51 , 72 ]. Afterwards, our null or PdΔζ knockout mutant was the first F 1 F O -ATPase inhibitor that showed a clear phenotypic difference between the wild-type and null mutant strains [ 42 , 44 , 50 ].…”

ATP Synthase Diseases of Mitochondrial Genetic Origin

“…This knowledge also enables to evaluate the observed pathogenic forms of mitochondrial morphology that are associated with these syndromes on the structural level, from the mutation at the molecular level to its associated consequences at the macroscopic scale of the organelle. The recent technical advances enabling the structural analysis of macromolecular complexes by cryo-EM (Kühlbrandt, 2014 ), the advent of complete structures of ATP synthases (Allegretti et al, 2015 ; Morales-Rios et al, 2015b ; Zhou et al, 2015 ; Hahn et al, 2016 ; Sobti et al, 2016 ; Guo et al, 2017 ) and the availability of genetically approachable systems like S. cerevisiae are just the first steps in these new shoes; they will considerably improve the comprehension of human diseases associated to defects in this key mitochondrial enzyme. We are still at the beginning of understanding these complex processes.…”

The Inhibitory Mechanism of the ζ Subunit of the F1FO-ATPase Nanomotor of Paracoccus denitrificans and Related α-Proteobacteria

“…Attempts to crystallize the PdF 1 -ATPase had resulted in non-diffracting or unstable PdF 1 -ATPase preparations that dissociate upon crystallization and produced diffracting crystals of a single ␣/␤ catalytic pair, thus lacking the ␥, ⑀, and subunits (18). In order to obtain a suitable structural model of the PdF 1 -complex, an initial homology model for the PdF 1 -ATPase was constructed using the most similar and complete crystallographic structure of the F 1 -ATPase available (Protein Data Bank entry 2WSS).…”