Functional Oligomeric Forms of Uncoupling Protein 2: Strong Evidence for Asymmetry in Protein and Lipid Bilayer Systems

Abstract

Stoichiometry of uncoupling proteins (UCPs) and their coexistence as functional monomeric and associated forms in lipid membranes remain intriguing open questions. In this study, tertiary and quaternary structures of UCP2 were analyzed experimentally and through molecular dynamics (MD) simulations. UCP2 was overexpressed in the inner membrane of Escherichia coli, then purified and reconstituted in lipid vesicles. Structure and proton transport function of UCP2 were characterized by circular dichroism (CD) spectroscopy and fluorescence methods. Findings suggest a tetrameric functional form for UCP2. MD simulations conclude that tetrameric UCP2 is a dimer of dimers, is more stable than its monomeric and dimeric forms, is asymmetrical and induces asymmetry in the membrane’s lipid structure, and a biphasic on–off switch between the dimeric units is its possible mode of transport. MD simulations also show that the water density inside the UCP2 monomer is asymmetric, with the cytoplasmic side having a higher water density and a wider radius. In contrast, the structurally comparable adenosine 5′-diphosphate (ADP)/adenosine 5′-triphosphate (ATP) carrier (AAC1) did not form tetramers, implying that tetramerization cannot be generalized to all mitochondrial carriers.

Molecular Dynamics Simulations of Mitochondrial Uncoupling Protein 2

“…These results are in accordance with the general structure analysis described in the previous sections, and promote UCP2 h structure as a potentially relevant structure for further MD simulation and mechanistic studies. We should mention here that the MD simulations of UCP2 based on ANT homology structure presented in Reference [37] agreed with the presented MD simulations. In particular, the constriction at the matrix side and opening of the cytosolic side of UCP2 protein had been observed as well, together with the low number density of water inside the protein cavity similar to the UCP2 h structure (Figures 5 and 6).…”

“…In contrast, the closely related UCP1 protein extracted using DPC and the structurally different detergent TX-100 remained physiologically active [20]. Interestingly, a recent study showing the oligomerization of UCP2 monomers did not describe differences between NMR and homology model structures [37].…”

Uncoupling Proteins and Regulated Proton Leak in Mitochondria

“…Consistent with SDS titration data, computational analysis of a UCP2 tetramer demonstrated that the dissociation energy of tetramer to dimer was ~20 kcal/mol lower than dissociation of dimer to monomer (tetramer to dimer: 60 kcal/mol; and dimer to monomer: 80 kcal/mol) [55]. Molecular dynamics (MD) simulations of UCP2 in POPC bilayer, also demonstrated a pseudosymmetrical structure for the tetramer in which the transport state of each dimeric pair open towards one side of the membrane was opposed by the other dimeric pair open on the other side of the membrane (either VVΛΛ or ΛΛVV) [55]. Furthermore, two salt-bridges were observed between the monomers within a dimer while no salt-bridge was observed between the dimers (Figure 3) [55].…”

“…These observations led to the proposition that the UCP tetramer is in fact a dimer of dimers, in which there is a loose binding interface between the dimers and a tight binding interface between the monomers within each pair of dimers (Figure 3) [11,28]. Consistent with SDS titration data, computational analysis of a UCP2 tetramer demonstrated that the dissociation energy of tetramer to dimer was ~20 kcal/mol lower than dissociation of dimer to monomer (tetramer to dimer: 60 kcal/mol; and dimer to monomer: 80 kcal/mol) [55]. Molecular dynamics (MD) simulations of UCP2 in POPC bilayer, also demonstrated a pseudosymmetrical structure for the tetramer in which the transport state of each dimeric pair open towards one side of the membrane was opposed by the other dimeric pair open on the other side of the membrane (either VVΛΛ or ΛΛVV) [55].…”

Assessing the Role of Lipids in the Molecular Mechanism of Membrane Proteins

“…By combining these two approaches, they can show that the serotonin receptor oligomeric state is correlated with PIP 2 availability. These research works and others [48,49] demonstrated that lipids can be part of the subunit interface, and directly stabilize the complex assembly. These findings also explain a number of functional studies that had observed a lipid-dependent modulation of function [7,50].…”