A Biophysical Study of the Ion Transport Mechanism in Uncoupling Protein 2 by Investigating the Role of Lysine Residues in its Matrix Network

ABSTRACT Uncoupling protein 2 (UCP2) is one of five UCP homologues found in the inner mitochondrial membrane that transports protons from the intermembrane space to the mitochondrial matrix. In turn, the proton motive force is dissipated and less ATP is produced in the mitochondria. UCP2 is proposed to influence insulin secretion in type II diabetes, and decrease the amount of reactive oxygen species produced in the mitochondria, however the detailed mechanism of ion (proton and anions) transport in UCP2 and other UCP homologues are not fully understood. Sequence alignment analysis performed on proteins in the mitochondrial carrier family (MCF) including UCPs, identified a matrix network of positively and negatively charged residues that were proposed to form salt bridges and mediate substrate translocation through the proteins. In this study, the positively charged lysine residues in the matrix network were investigated for their influence on the proton transport and nucleotide binding activity of UCP2. For this reason, four UCP2 mutants: K38Q, K141Q, K239Q, and K38Q/K239Q (double mutant) and native proteins were expressed in bacterial membranes. After which the conformation of the purified proteins was analyzed with far-UV circular dichroism (CD). Finally, a fluorescence-based assay was used to study the proton transport and nucleotide binding properties of the proteins. The overall conformations of the proteins were α-helical but the shifts in negative ellipticity at 208 nm and 222 nm observed for the mutants inferred a change in the helical packing of these proteins compared to the wild type. In addition, the mutant proteins had proton transport rates that were 35% (K38Q and K239Q) and 65% (K141Q and double mutant) less than the native UCP2. In the presence of ATP, the proton transport rates of the mutant proteins decreased by 3-6% except for K38Q that had a 38% decrease in proton transport activity. In summary, these results revealed that the positively charged lysine residues in the matrix network could participate in a salt bridge interaction that regulates the degree of helical packing, the ion transport activity and nucleotide binding properties in UCP2

Cardiolipin Prevents Membrane Translocation and Permeabilization by Daptomycin

This research was originally published in Journal of Biological Chemistry. Zhang, T., Muraih, J. K., Tishbi, N., Herskowitz, J., Victor, R. L., Silverman, J., … Mintzer, E. (2014). Cardiolipin Prevents Membrane Translocation and Permeabilization by Daptomycin. Journal of Biological Chemistry, 289(17), 11584–11591. © the American Society for Biochemistry and Molecular Biology." Available here: https://doi.org/10.1074/jbc.M114.554444Daptomycin is an acidic lipopeptide antibiotic that, in the presence of calcium, forms oligomeric pores on membranes containing phosphatidylglycerol. It is clinically used against various Gram-positive bacteria such as Staphylococcus aureus and Enterococcus species. Genetic studies have indicated that an increased content of cardiolipin in the bacterial membrane may contribute to bacterial resistance against the drug. Here, we used a liposome model to demonstrate that cardiolipin directly inhibits membrane permeabilization by daptomycin. When cardiolipin is added at molar fractions of 10 or 20% to membranes containing phosphatidylglycerol, daptomycin no longer forms pores or translocates to the inner membrane leaflet. Under the same conditions, daptomycin continues to form oligomers; however, these oligomers contain only close to four subunits, which is approximately half as many as observed on membranes without cardiolipin. The collective findings lead us to propose that a daptomycin pore consists of two aligned tetramers in opposite leaflets and that cardiolipin prevents the translocation of tetramers to the inner leaflet, thereby forestalling the formation of complete, octameric pores. Our findings suggest a possible mechanism by which cardiolipin may mediate resistance to daptomycin, and they provide new insights into the action mode of this important antibiotic

Biphasic Proton Transport Mechanism for Uncoupling Proteins.

It has been suggested that uncoupling proteins (UCPs) transport protons via interconversion between two conformational states: one in the cytoplasmic state and the other in the matrix state . Matrix and cytoplasmic salt-bridge networks are key controllers of these states. This study proposes a mechanism for proton transport in tetrameric UCP2, with focus on the role of the matrix network. Eleven mutants were prepared to disrupt (K → Q or D → N mutations) or alter (K → D and D → K mutations) the salt-bridges in the matrix network. Proteins were recombinantly expressed in Escherichia coli membrane, reconstituted in model lipid membranes, and their structures and functions were analyzed by gel electrophoresis, circular dichroism spectroscopy, fluorescence assays, as well as molecular dynamics simulations. It is shown that the UCP2 matrix network contains five salt-bridges (rather than the previously reported three), and the matrix network can regulate the proton transport by holding the protein\u27s transmembrane helices in close proximity, limiting the movement of the activator fatty acid(s). A biphasic two-state molecular model is proposed for proton transport in tetrameric (a dimer of stable dimers) UCP2, in which all the monomers are functional, and monomers in each dimer are in the same transport mode. Purine nucleotide (e.g., ATP) can occlude the internal pore of the monomeric units of UCP tetramers via interacting with positive residues at or in the proximity of the matrix network (K38, K141, K239, R88, R185, and R279) and prevent switching between cytoplasmic and matrix states, thus inhibiting the proton transport. This study provides new insights into the mechanism of proton transport and regulation in UCPs

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

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\u27s 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\u27-diphosphate (ADP)/adenosine 5\u27-triphosphate (ATP) carrier (AAC1) did not form tetramers, implying that tetramerization cannot be generalized to all mitochondrial carriers