Amputation of a C-terminal helix of the γ subunit increases ATP-hydrolysis activity of cyanobacterial F1 ATP synthase

Abstract

F is a soluble part of FF-ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The γ subunit, which is the rotary shaft of F motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of γ assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of αβ hexamer of the F molecule. Cyanobacterial and chloroplast γ subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial γ. To understand the structure-function relationship of the γ subunit, we prepared a mutant F-ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the γ subunit is split into N-terminal α-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type γ. Contrary to our expectation, the complexes containing the split γ subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the γ subunit. We demonstrate that insertion of the nick between two helices of the γ subunit imparts resistance to ADP inhibition, and the C-terminal α-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F-ATP synthase.

The phototroph-specific β-hairpin structure of the γ subunit of FoF1-ATP synthase is important for efficient ATP synthesis of cyanobacteria

“…These results strongly suggested that the interaction between the DELSEED loop of the β subunit and the two amino acids located at the turn of the β-hairpin structure is sufficient to induce MgADP inhibition upon ATP hydrolysis. This model was supported by our studies using recombinant α 3 β 3 γ derived from T. elongatus with nick insertion into the proximal region of the β-hairpin structure (between V 222 and T 223 ) and cross-linking experiments using disulfide bond formation between the central stalk and the β-hairpin structure within the γ subunit ( 31 , 32 ).…”

“…For immunoblotting, separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Immun-Blot, Bio-Rad). Antibodies against the c subunit were obtained from Agrisera, and those against the β subunit were as described in the literature ( 31 ). Chemiluminescence was detected using horseradish peroxidase–conjugated secondary antibodies and ECL Prime (Life Technologies) and visualized on a LAS 3000 mini instrument (GE Healthcare).…”

“…ATP hydrolysis activity was measured using an ATP-regenerating system as described ( 31 ), with some modifications. The assay was conducted at 30 °C.…”