The ATP-bound State of the Uncoupling Protein 1 (UCP1) from Molecular Simulations
The uncoupling protein 1 (UCP1) dissipates the transmembrane (TM) proton gradient in the inner mitochondrial membrane (IMM) by leaking protons across the membrane, producing heat in the process. Such non-shivering production of heat in brown adipose tissue can combat obesity-related diseases. UCP1 associated proton leak is activated by free fatty acids and inhibited by purine nucleotides. The mechanism of proton leak remains unknown, in part due to the unavailability of high-resolution structures of the protein. As a result, the binding site of the activators (fatty acids) and inhibitors (nucleotides) is unknown. Using molecular dynamics simulations, we generate a conformational ensemble of UCP1. Using Metadynamics-based free energy calculations, we converge on the most likely ATP-bound conformation of UCP1. Our conformational ensemble provides a molecular basis of a breadth of prior biochemical data available for UCP1. Based on the simulations, we make the following testable predictions about the mechanisms of activation of proton leak and proton leak inhibition by ATP: (1) R277 plays the dual role of stabilising ATP at the binding site for inhibition, and acting as a proton surrogate for D28 in the absence of a proton during proton transport (2) the binding of ATP to UCP1 is mediated by residues R84, R92, R183, and S88 (3) R92 shuttles ATP from the E191-R92 gate in the inter-membrane space to the nucleotide binding site, and serves to increase ATP affinity (4) ATP can inhibit proton leak by controlling the ionisation states of matrix facing lysine residues such as K269 and K56 and (5) fatty acids can bind to UCP1 from the IMM either via the cavity between TM1 and TM2 or between TM5 and TM6. Our simulations set the platform for future investigations into the proton transport and inhibition mechanisms of UCP1.