Biphasic Proton Transport Mechanism for Uncoupling Proteins

Abstract

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.

Investigating the Broad Matrix-Gate Network in the Mitochondrial ADP/ATP Carrier through Molecular Dynamics Simulations

“…The difference between the m-gate networks with and without presence of CATR was also highlighted in earlier MD simulation studies on AAC [ 13 , 19 ]. A recent study on the uncoupling protein UCP2 also demonstrated the engagement of the three triplet 79 residues (R88, R185 and R279) in the broad matrix network [ 20 ]. Therefore, results from different groups consistently suggest that these non-motif basic residues are integral part of the broad matrix network.…”

Uncoupling Proteins and Regulated Proton Leak in Mitochondria

“…Furthermore, mutagenetic and functional analysis of UCPs by different researchers and different methods provide important insights about fatty acid-UCP interactions, as well as residues that are involved in UCP-mediated transport. Table 1 shows the residues that are involved in proton transport regulation along with the major findings of related studies [4,28,53,59,60,[62][63][64][65][66][67][68]. It is widely accepted that proton flux by UCPs is activated by fatty acids [28,61].…”

“…All four proposed fatty-acid-mediated transport models involve mechanisms of proton transport through monomeric units of UCPs. Recently, a more complex Biphasic Two-State molecular model has been proposed for proton transport through oligomeric states of UCPs [55,68]. This model is based on a thorough comparative structural and functional analysis of tetrameric UCP2 and several mutants in model lipid bilayers [55,68].…”

“…Concurrently, conformational changes of one dimeric pair can influence the active/inactive conformation of the other pair. Considering the loose interface between the two dimeric pairs [55], it has been suggested that dimerization of dimers might be an indication of a higher level of regulation of proton transport by UCPs, as compared with proton transport through monomers [68] (Figure 6). None of these models are universally accepted and continue to be debated [50,69].…”

“…A suggested inhibitory mechanism of GDP is that it can bind inside the translocation channel of the protein and allosterically displace fatty acid from its binding site [53]. A recent study on the structure and function of tetrameric UCP2 provided evidence that purine nucleotides such as ATP can bind to positive residue at/close to the matrix network (K38, K141, K239, R88, R185, R279) in each subunit and occlude the internal channel thus inhibiting the protein from conformational changes required for proton transport [68].…”