Experimental and Computational Evidence for Self-Assembly of a Functional Form of Mitochondrial Uncoupling Protein 2 in Lipid Bilayers

Uncoupling proteins (UCPs) are members of the mitochondrial carrier family (MCF) that transport protons across the inner mitochondrial membrane, thereby uncoupling electron transport from ATP synthesis. The stoichiometry of UCPs, and the possibility that they form homo-oligomers within lipid membranes remain un-resolved. In the current study, the tertiary structure of UCP2 was analyzed experimentally and using molecular dynamics (MD) simulations. Recombinantly expressed UCP2 was purified both as a pure monomer and as a mixture of monomers, dimers and tetramers. Both preparations were reconstituted in phosphatidylcholine (PC) vesicles. Gel electrophoresis, circular dichroism spectroscopy and fluorescence methods were used to characterize the protein. UCP2 only showed stable tetrameric forms in lipid bilayers. MD simulations using PC lipids and principal component analysis support the experimental results and provided insights into the nature of noncovalent interactions in oligomeric UCP2. MD simulations indicate that UCP2 tetramers are asym-metric dimers of dimers, in which the interactions between the monomers forming the dimer are stronger than the interactions between the dimers within the tetramer. It is also shown that UCP2 has a specific tendency to form functional tetramers in lipid bilayers, capable of proton transport. The asymmetric nature of the UCP2 tetramer could act as a scaffold for regulating the activity of the monomeric units through cooperative intercommunication between these subunits. Under similar experimental conditions, the structurally comparable ADP/ATP carrier protein did not form tetramers in PC vesicles, implying that spontaneous tetramerization cannot be generalized to all MCF members.