No 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.

The exopolysaccharide Psl contributes to biofilm structure and antibiotic tolerance and may play a role in the failure to eradicate Pseudomonas aeruginosa from cystic fibrosis (CF) airways. The study objective was to determine whether there were any differences in Psl in P. aeruginosa isolates that were successfully eradicated compared to those that persisted, despite inhaled tobramycin treatment, in children with CF. Initial P. aeruginosa isolates were collected from children with CF undergoing eradication treatment, grown as biofilms and labeled with 3 anti-Psl monoclonal antibodies (Cam003/Psl0096, WapR001, WapR016) before confocal microscopy visualization. When grown as biofilms, P. aeruginosa isolates from children who failed antibiotic eradication therapy, had significantly increased Psl0096 binding compared to isolates from those who cleared P. aeruginosa. This was confirmed in P. aeruginosa isolates from the SickKids Eradication Cohort as well as the Early Pseudomonas Infection Control (EPIC) trial. Increased anti-Psl antibody binding was associated with bacterial aggregation and tobramycin tolerance. The biofilm matrix represents a potential therapeutic target to improve P. aeruginosa eradication treatment.

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's 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.

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.