Three-Pronged Computational Approach to Predict Regulatory Ligands of Cytochrome C Oxidase

Abstract

A crystallographically-defined bile acid binding site has been identified in the membrane domain of mammalian and Rhodobacter sphaeroides cytochrome c oxidase (RsCcO). Previous studies indicate that several amphipathic molecules including detergents, fatty acids, and porphyrins also bind to this site and alter K-pathway dependent electron transfer between heme cofactors. Current studies are aimed at identifying physiological ligands specific for this site using several computational approaches, including: ROCS comparison of ligand shape and electrostatics, SimSite3D analysis of similarity to ligand binding sites in the Protein Data Bank, and SLIDE screening of small molecules by docking. Together, the results suggest several steroids, adenine and guanine deoxyribonucleotides, NAD+, FAD, and phosphorylated isoprenes as top candidates for interacting at this site, along with bile acids and porphyrins. Deoxyriboside-containing ligands are all predicted to bind to this site by making key protein contacts with Pro315, His96, Ser98, Glu101, and Thr105, as seen in bile acid binding. Invitro oxygen consumption assays support some of these predicted interactions. In the wildtype RsCcO, the steroidal antibiotic fusidic acid and T3 thyroid hormone inhibit the enzyme, while in the RsCcO E101A mutant, fusidic acid has a stimulatory effect and an ATP analog and T3 thyroid hormone inhibit activity. Cytochrome c titration assays indicate that nucleotides also inhibit the E101A mutant enzyme at lower cytochrome c concentrations. The confirmed prediction of new ligands suggests that this three-pronged computational approach may be applied to identify native ligands in sites occupied by detergents or crystallographic additives. (supported by NIH GM26916 to SF-M)