Study of the Yeast Vacuolar-type ATPase by Electron Cryomicroscopy

Vacuolar-type ATPases (V-ATPases) are rotary enzymes that use energy from hydrolysis of adenosine triphosphate (ATP) to pump protons across membranes and control the pH of many intracellular compartments. ATP hydrolysis in the soluble catalytic region of the enzyme is coupled to proton translocation through the membrane-bound region by rotation of a rotor subcomplex. Studies of ATP synthases, V-ATPases, and vacuolar/archaeal ATPases (V/A-ATPases) have suggested that flexibility is necessary for the catalytic mechanism of rotary ATPases but the structures of different rotational states have never been observed experimentally. V-ATPases also serve an important role in the immune system and are targeted by intracellular pathogens. Legionella pneumophila, the causative agent of Legionnaires' Disease, secretes a protein effector called SidK that binds and inhibits V-ATPases, preventing acidification of lysosomes and promoting survival of the bacterium inside macrophages. The mechanism of V-ATPase inhibition by SidK is not known. The structure of V-ATPases can be analyzed by single particle electron cryomicroscopy (cryo-EM), an imaging technique involving the identification of tens to hundreds of thousands of individual proteins in micrographs. A partially-automated image selection program was developed to increase particle image identification throughput. A method was also developed to correct for magnification anisotropy that was identified in our images. Cryo-EM maps were obtained for structures of three rotational states of the V-ATPase from the yeast Saccharomyces cerevisiae. The three different maps reveal the conformational changes that occur to couple rotation in the soluble catalytic region to the symmetry-mismatched membrane-bound proton-translocating region. The structures of these states provide direct evidence that deformation during rotation enables the smooth transmission of power through rotary ATPases. To investigate the molecular mechanism of V-ATPase inhibition by SidK, we determined structures of the V-ATPase:SidK complex by cryo-EM. The cryo-EM maps reveal SidK to be an elongated protein composed of a structured region that interacts with the soluble catalytic region of V-ATPase and a flexible region of unknown function. The structures suggest a novel inhibition site for V-ATPases and provides evidence that the activity of these proton pumps is linked to the flexibility of the A-subunit.Ph.D