Experimental and Computational Evidence for Self-Assembly of Mitochondrial UCP2 in Lipid Bilayers

Abstract

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 unresolved. 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 asymmetric 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/anti-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.

Polyunsaturated Fatty Acid Modulates Membrane-Bound Monomeric α-Synuclein by Modulating Membrane Microenvironment through Preferential Interactions

“…All simulations were performed with all-atom CHARMM36m force field parameters for protein, CHARMM36 parameter set for lipids and salt, and CHARMM-modified TIP3P model for water. − The same set of parameters has been used in many recent computer simulation studies of membrane–protein systems. − The CHARMM36m force field involves refinement of CHARMM36 force field, with improved accuracy for generating conformational ensembles for intrinsically disordered proteins …”