The role of matrix-oriented salt-bridge on the proton transport in a multimeric uncoupling protein 2

Uncoupling proteins (UCPs) are members of the mitochondrial carrier superfamily (MCF) that transport protons across the inner mitochondrial membrane, thereby uncoupling respiration from ATP synthesis. The proton transport mechanism in UCPs is not fully understood. It has been suggested that members of MCF possess matrix and cytoplasmic salt-bridge networks, which could control substrate transport via alternative conformational changes. In the current study, the role of the matrix-facing salt-bridge network in proton transport function of UCP2 and its molecular state in membranes were analyzed. The role of the matrix network was evaluated by introducing single and double mutations of positively-charged lysine residues (K38Q, K141Q, K239Q, and K38Q/K239Q) into UCP2. Proteins were expressed in E. coli membranes and reconstituted in phosphatidylcholine vesicles. CD spectroscopy and fluorescence methods were employed to characterize protein structure and function, respectively. Proton transport was significantly increased in single mutants compared to the wild-type protein but did not change significantly in the double mutant, suggesting a regulatory role for the matrix network. Monomeric UCP2 self-associated into a tetrameric functional form after reconstitution in lipid vesicles. This result was further supported by molecular dynamics (MD) simulations. The molecular forms of UCP2 in liposomes were also compared to those of another conformationally relevant protein, ADP/ATP carrier. In contrast to UCP2, ADP/ATP carrier did not form tetramers in lipid membranes. Overall, tetrameric forms of UCP2 transported protons in lipid membranes and this transport was regulated by the matrix salt-bridge network.