title: Structure-function relationships in UCP1, UCP2 and chimeras: EPR analysis and retinoic acid activation of UCP2.
abstract: Uncoupling proteins (UCPs) are composed of three repeated domains of approximately 100 amino acids each. We have used chimeras of UCP1 and UCP2, and electron paramagnetic resonance (EPR), to investigate domain specific properties of these UCPs. Questions include: are the effects of nucleotide binding on proton transport solely mediated by amino acids in the third C-terminal domain, and are the amino acids in the first two domains involved in retinoic or fatty acid activation? We first confirmed that our reconstitution system produced UCP1 that exhibited known properties, such as activation by fatty acids and inhibition of proton transport by purine nucleotides. Our results confirm the observations reported for recombinant yeast that retinoic acid, but not fatty acids known to activate UCP1, activates proton transport by UCP2 and that this activation is insensitive to nucleotide inhibition. We constructed chimeras in which the last domains of UCP1 or UCP2 were switched and tested for activation by fatty acids or retinoic acid and inhibition by nucleotides. U1U2 is composed of mUCP1 (amino acids 1-198) and hUCP2 (amino acids 211-309). Fatty acids activated proton transport of U1U2 and GTP mediated inhibition. In the other chimeric construct U2U1, hUCP2 (amino acids 1-210) and mUCP1 (amino acids 199-307), retinoic acid still acted as an activator, but no inhibition was observed with GTP. Using EPR, a method well suited to the analysis of the structure of membrane proteins such as UCPs, we confirmed that UCP2 binds nucleotides. The EPR data show large structural changes in UCP1 and UCP2 on exposure to ATP, implying that a putative nucleotide-binding site is present on UCP2. EPR analysis also demonstrated changes in conformation of UCP1/UCP2 chimeras following exposure to purine nucleotides. These data demonstrate that a nucleotide-binding site is present in the C-terminal domain of UCP2. This domain was able to inhibit proton transport only when fused to the N-terminal part of UCP1 (chimera U1U2). Thus, residues involved in nucleotide inhibition of proton transport are located in the two first carrier motifs of UCP1. While these results are consistent with previously reported effects of the C-terminal domain on nucleotide binding, they also demonstrate that interactions with the N-terminal domains are necessary to inhibit proton transport. Finally, the results suggest that proteins such as UCP2 may transport protons even though they are not responsible for basal or cold-induced thermogenesis.
authors.affiliation: Departments of Pediatrics, Section of Neurobiology Physiology and Behavior, Rowe Program in Human Genetics, and Department of Biological Chemistry, School of Medicine, University of California, Davis, USA.
authors.forename: N
authors.initials: N
authors.lastname: Chomiki
authors.forename: J C
authors.initials: JC
authors.lastname: Voss
authors.forename: C H
authors.initials: CH
authors.lastname: Warden
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D002846:Chromatography, Affinity; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000494:pharmacology; D006801:Humans; D007473:Ion Channels; D017136:Ion Transport / Q000187:drug effects; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D026901:Membrane Transport Proteins; D051379:Mice; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D017494:Proton Pumps / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D011685:Purine Nucleotides / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology*; D000071256:Uncoupling Protein 1; D000071246:Uncoupling Protein 2
chemical_list: D002352:Carrier Proteins; D005227:Fatty Acids; D007473:Ion Channels; D008081:Liposomes; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; D011506:Proteins; D017494:Proton Pumps; D011685:Purine Nucleotides; D011993:Recombinant Fusion Proteins; C000604371:UCP1 protein, human; C000604389:UCP2 protein, human; C000604372:Ucp1 protein, mouse; C000604390:Ucp2 protein, mouse; D000071256:Uncoupling Protein 1; D000071246:Uncoupling Protein 2; D014212:Tretinoin