The structural and functional role of the gamma-epsilon rotor in Escherichia coli FoF1 ATP synthase.

摘要

The gamma-epsilon-c subunits of the Escherichia coli FOF1 ATP synthase make up the rotor assembly, which couples proton transport to ATP catalysis. Previous EPR and functional studies from our laboratory suggest that interactions in the gamma-epsilon and gamma- c interfaces play important roles in efficient coupling. To further investigate the structure and dynamics of the interface, we have used the site-directed spin labeling strategy of EPR spectroscopy to define the structure of epsilon subunit in the gamma-epsilon-c subunit interface. EPR spectra of isolated epsilon subunits with spin labels at single-cysteine substitutions from epsilonE29C to epsilonA44C show an alternating mobility indicating a beta-strand secondary structure. When bound to F1 complex, spectra of spin-labeled epsilonP40C-epsilonT43C show large decreases of mobility, which are likely due to tertiary contacts with the gamma subunit. The epsilonL42C mutant subunit binds to the F 1 complex with lower affinity indicating that the hydrophobic interaction contributes significant amount of binding energy in gamma-epsilon interface. The EPR spectra of spin labeled epsilon29, epsilon33, epsilon35, epsilon36, epsilon37 and epsilon39 subunit-F1 complexes demonstrate large mobility decreases when F1 is reconstituted with the membranous FO sector, suggesting that these residues directly interact with subunit c. Single-cysteine mutations introduced in the epsilon-c interface do not disrupt energy coupling between FO and F1, implying that the gamma-c interface is the main channel for the energy coupling mechanism and one role of epsilon is to strengthen the gamma-c interaction and coordinate conformations of gamma and c subunits for optimal coupling efficiency.;Deuterium-Hydrogen exchange and X-ray footprinting methods were used to explore the solvent-accessibility changes of the rotor and rotor-stator interface regions in the presence and absence of ATP. However, due to the problem of deuterium-back-exchange, solvent accessibility map of the gamma-epsilon rotor could not be established based on the deuterium-hydrogen exchange method. From X-ray footprinting analysis, the nucleotide-dependent changes of oxidative rates of gammaMet178 and betaMet379 suggest the possible important contacts between gamma and alpha3beta3 hexamer during rotation. Decreases of oxidative rates of epsilonMet15, epsilonMet49, and epsilonPhe61 in the presence of ATP provide evidence for the nucleotide-dependent movement of C-terminal helices of epsilon subunit.