Atomic structure of kidney V-ATPase

NUS scientist has revealed the high-resolution atomic structure of the mammalian kidney vacuolar type ATPase, and discovered a novel binding partner for this complex molecule that is crucial for maintaining the acidity of urine.

Vacuolar type ATPases (V-ATPases) are important molecular machines found in numerous cell types because they act as a special kind of transporter to help move protons across a cell membrane. This is similar to how mechanical pumps move water across a dam. Due to their importance in maintaining the acidity or basicity of various cellular and extracellular compartments, dysfunction of V-ATPases results in diseases associated with the kidney (distal renal tubule acidosis), bone (osteopetrosis), brain (neurodegeneration) and also cancer.

V-ATPase is a massive rotary molecular machine found in our body made up of 16 different unique components. Together, these components are about a megadalton in mass. A dalton is the unit mass commonly used to express the mass of atomic-scale objects. As these components differ between the V-ATPases found in different organs and tissues, it makes mechanistic studies of them highly difficult.

To better understand the structure of this molecular machine, Assistant Professor TAN Yong Zi from the Department of Biological Sciences, National University of Singapore in collaboration with the laboratory of Professor John L. RUBINSTEIN from The Hospital of Sick Children (Canada) sought to obtain high purity samples of V-ATPase from kidney tissue. The method they used involved using a bacterial toxin known as SidK that is able to specifically bind itself to mammalian V-ATPase. This allowed the researchers to extract highly pure samples of this molecular machine which they imaged with a state-of-the-art cryogenic electron microscope. Using a computational process, the scientists were able to provide the first high resolution structure of a mammalian kidney V-ATPase in three different states of rotation.

Prof Tan said, “The cryogenic electron microscopy is able to resolve multiple states of a protein molecule from a single sample. This allows us to build a dynamic view of any protein complex using real data. In our study, we were able to see all the three major rotational states of the V-ATPase, which when morphed together shows its complete catalytic cycle.”

An additional surprise came along when the scientists found the presence of a protein called mEAK-7 bound to a tiny fraction of the V-ATPase.  This particular protein is known to be involved in nutrient signaling and could prove to be a critical link on how the cell regulates its feeding depending on the contents of its lysosome (the cell’s stomach).

 “These high resolution structures could potentially pave the way for better mechanistic understanding of this complex molecule, and for developing drugs against the various diseases that afflict patients due to V-ATPase mutations,” added Prof Tan.

Tan YZ; Abbas YM; Wu JZ; Wu D; Keon KA; Hesketh GG; Bueler SA; Gingras AC; Robinson CV; Grinstein S; Rubinstein JL*, “CryoEM of endogenous mammalian V-ATPase interacting with the TLDc protein mEAK-7” LIFE SCIENCE ALLIANCE Volume: 5 Issue: 11 Article Number: e202201527 DOI: 10.26508/lsa.202201527 Published: 2022.