Structure of the γ–ε complex of cyanobacterial F1-ATPase reveals a suppression mechanism of the γ subunit on ATP hydrolysis in phototrophs

Abstract

F-ATPase forms the membrane-associated segment of FF-ATP synthase - the fundamental enzyme complex in cellular bioenergetics for ATP hydrolysis and synthesis. Here, we report a crystal structure of the central F subcomplex, consisting of the rotary shaft γ subunit and the inhibitory ε subunit, from the photosynthetic cyanobacterium BP-1, at 1.98 Å resolution. In contrast with their homologous bacterial and mitochondrial counterparts, the γ subunits of photosynthetic organisms harbour a unique insertion of 35-40 amino acids. Our structural data reveal that this region forms a β-hairpin structure along the central stalk. We identified numerous critical hydrogen bonds and electrostatic interactions between residues in the hairpin and the rest of the γ subunit. To elaborate the critical function of this β-hairpin in inhibiting ATP hydrolysis, the corresponding domain was deleted in the cyanobacterial F subcomplex. Biochemical analyses of the corresponding αβγ complex confirm that the clinch of the hairpin structure plays a critical role and accounts for a significant interaction in the αβ complex to induce ADP inhibition during ATP hydrolysis. In addition, we found that truncating the β-hairpin insertion structure resulted in a marked impairment of the interaction with the ε subunit, which binds to the opposite side of the γ subunit from the β-hairpin structure. Combined with structural analyses, our work provides experimental evidence supporting the molecular principle of how the insertion region of the γ subunit suppresses F rotation during ATP hydrolysis.

The structure of the catalytic domain of the ATP synthase from Mycobacterium smegmatis is a target for developing antitubercular drugs

“…S11; see SI Appendix, Fig. S15) that wedges between the β-subunit and the central stalk thereby blocking the rotation of the γ-subunit and preventing futile ATP hydrolysis (43). With daylight and a rising proton motive force, the synthetic activity of the enzyme is restored by reduction of the disulfide bond by thioredoxin.…”

The N-terminal region of the ε subunit from cyanobacterial ATP synthase alone can inhibit ATPase activity

“…There is therefore a possibility that the ⑀ subunit that lacks CTD did not sufficiently associate with the ␥ subunit of chloroplast ATP synthase in their experimental conditions (30) and did not inhibit F 1 -ATPase activity very well. Recently, the whole molecular structure of chloroplast ATP synthase was determined by cryo-EM (43), and our group also determined the X-ray crystal structure of the cyanobacterial ␥-⑀ subcomplex (16). In both structures, CTD of the ⑀ subunit showed a retracted form, although CTDs of the ⑀ subunits from Escherichia coli and Geobacillus stearothermophilus (formerly known as thermophilic Bacillus PS3) were extended in the crystal structures (44,45).…”

“…The ␥ subunits of chloroplast-type (cyanobacteria and chloroplast-type) ATP synthase equip the insertion region (Fig. 7), which also functions to inhibit ATP hydrolysis activity (16). We therefore investigated the involvement of this region with the ⑀-inhibition of cyanobacterial ATP synthase in Fig.…”

“…Therefore, both the insertion region of the ␥ subunit of cyanobacterial ATP syn- The ⑀-inhibition of cyanobacterial F 1 -ATPase thase and ⑀-NTD must be involved in the regulation of ATP hydrolysis by the tight interaction between the ␥ and ⑀ subunits. In addition, the ⑀ subunit may affect the conformation of the insertion region of the ␥ subunit, which was found to be a ␤-hairpin structure (16,43), to control the ATP hydrolysis activity in cyanobacteria and have an impact on the redox state of the ␥ subunit from chloroplast ATP synthase as well (48).…”

“…One of the most well-known regulatory mechanisms is MgADP inhibition (ADP inhibition), which is induced by occupation of the catalytic site with MgADP and prevents the ATP hydrolysis reaction (14). The ␥ subunits of chloroplast-type ATP synthases, including the cyanobacterial one, possess an insertion region composed of 30 -40 amino acids between the Rosmann-fold domain and the C-terminal domain (CTD) 2 of ␣-helices, which is not observed in the ␥ subunit of other F 1 ; this region has a function in regulating F 1 -ATPase (15,16). In addition, the ␥ subunit of the chloroplast ATP synthase has an additional nine-amino acid insertion containing a pair of Cys residues at this insertion region, and this Cys pair is key for the redox control by thioredoxin (17,18).…”