A proteomic approach to resolving the binding sites for tentoxin in plastid CF1-ATPase

Abstract

F0F1-ATPase (ATP synthase) is a central enzyme in energy conversion in chloroplasts, mitochondria and bacteria. The main reaction catalyzed is ATP formation from ADP and Pi, using energy derived from a transmembrane elctrochemical potential gradient. Tentoxin, a fungal cyclic tetrapeptide, affects the soluble component (CF1) of chloroplast ATPase, inhibiting photophosphorylation at low concentrations and stimulating it at higher ones. The inhibitory site of tentoxin is known to be associated with the a and b subunits of CF1-ATPase with, codon 83 of the b subunit being a molecular-genetic hot spot determining sensitivity to the toxin in various higher plants and algae. Considerable sequence and structural homology exists between the a and b subunits of mitochondrial and chloroplast F0F1-ATPase. The resolved structure for an a3b3g fragment of F1-ATPase of bovine mitochondria, containing stoichiometric amounts of bound nucleotides, is present in the public structural database. Using LigIn software for molecular docking and LPC software for analysis, we located and analyzed theoretical inhibitory and stimulatory binding sites for tentoxin in this structure. Complementarity to tentoxin at these sites is sensitive to the nucleotide occupancy state of the b subunit. The conclusions from modeling the theoretical binding sites for tentoxin in the resolved a3b3g crystal structure were applied to modeling analogous sites in unresolved plastid CF1-ATPase of Chlamydomonas. The residues most likely involved in the direct binding of tentoxin in CF1-ATPase are so identified. These residues are prime targets for modification to control the tentoxin response in plants.