Conserved glycine residues in the P-loop of ATP synthases form a doorframe for nucleotide entrance

The phosphate binding loop (GXXXXGKT(S)) is conserved in several mononucleotide-binding proteins with similar three-dimensional structures. Although variations in other amino acids have been noted, the first glycine and glycine-lysine residues are highly conserved in all enzymes, whose role is yet to be understood. Alanine substitutions for critically positioned glycines—G234, G237, and G239—were generated for the catalytic A-subunit of A-ATP synthase from Pyrococcus horikoshii OT3, and their crystal structures were determined. They showed altered conformation for the phosphate binding loop, with G234A and G237A becoming flat and with G239A taking an intermediate conformation, resulting in the active-site region being closed to nucleotide entry. Furthermore, the essential amino acids S238 and K240, which normally interact with the nucleotide, become inaccessible. These mutant structures demonstrate the role of the strictly conserved glycine residues in guarding the active-site region for nucleotide entrance in archaea-type ATP synthases.     

Nucleotide Binding States of Subunit A of the A-ATP Synthase and the Implication of P-Loop Switch in Evolution

The crystal structures of the nucleotide-empty (A_E), 5′-adenylyl-β,γ-imidodiphosphate (A_PNP)-bound, and ADP (A_DP)-bound forms of the catalytic A subunit of the energy producer A₁A_O ATP synthase from Pyrococcus horikoshii OT3 have been solved at 2.47 Å and 2.4 Å resolutions. The structures provide novel features of nucleotide binding and depict the residues involved in the catalysis of the A subunit. In the A_E form, the phosphate analog SO₄²⁻ binds, via a water molecule, to the phosphate binding loop (P-loop) residue Ser238, which is also involved in the phosphate binding of ADP and 5′-adenylyl-β,γ-imidodiphosphate. Together with amino acids Gly234 and Phe236, the serine residue stabilizes the arched P-loop conformation of subunit A, as shown by the 2.4-Å structure of the mutant protein S238A in which the P-loop flips into a relaxed state, comparable to the one in catalytic β subunits of F₁F_O ATP synthases. Superposition of the existing P-loop structures of ATPases emphasizes the unique P-loop in subunit A, which is also discussed in the light of an evolutionary P-loop switch in related A₁A_O ATP synthases, F₁F_O ATP synthases, and vacuolar ATPases and implicates diverse catalytic mechanisms inside these biological motors.