Crystal Structures Of Yeast Mitochondrial ATPase with Uncoupling Mutations

Abstract

Yeast mitochondrial ATP synthase is a transmembrane protein responsible for synthesis of more than 90% of ATP under aerobic conditions. The water soluble portion of ATP synthase, F1, is composed of five subunits with stoichiometry α3β3γδe and has a combined molecular weight of 360 kDa. The three active sites of ATPase are formed at the interfaces between alternating α- and β-subunits. ATPase is capable of efficient ATP hydrolysis, accompanied by rotation of central stalk subunits, γδe, within the α3β3 core and is capable of ATP synthesis if central subunits are forced to rotate in the opposite direction. A number of mutations in ATP synthase have been identified that result in the uncoupling of catalytic function and proton flow across the mitochondrial membrane. These uncoupling mutations cluster at the interface between γ-subunit and α3β3 catalytic core of ATPase. In this work, four X-ray crystal structures of ATPase with single amino acid substitutions αN67I, αF405S, βV279F, and γI270T were solved at resolutions ranging from 3.2 A to 2.74 A. This study will present a structural comparison of the mutant structures with the wild type structures to understand the mechanism of coupling. However, the crystal structures likely represent the ground state of catalytic reaction cycle while the mutations may result in notable distortions of enzyme structure during other stages of catalytic cycle. Additionally, the uncoupling of the ATPase may be caused by changes in the energy of interaction between the portions that are rotating in the molecular machine thereby altering transition to the higher energy states. Structural based hypotheses are presented to explain the role of these residues in the coupling of the enzyme. Supported by NIH R01GM0662223