Identification by site-directed mutagenesis of three arginines in uncoupling protein that are essential for nucleotide binding and inhibition.
Primary regulation of uncoupling protein is mediated by purine nucleotides, which bind to the protein and allosterically inhibit fatty acid-induced proton transport. To gain increased understanding of nucleotide regulation, we evaluated the role of basic amino acid residues using site-directed mutagenesis. Mutant and wild-type proteins were expressed in yeast, purified, and reconstituted into liposomes. We studied nucleotide binding as well as inhibition of fatty acid-induced proton transport in wild-type and six mutant uncoupling proteins. None of the mutations interfered with proton transport. Two lysine mutants and a histidine mutant had no effect on nucleotide binding or inhibition. Arg83 and Arg182 mutants completely lost both the ability to bind nucleotides and nucleotide inhibition. Surprisingly, the Arg276 mutant exhibited normal nucleotide binding, but completely lost nucleotide inhibition. To account for this dissociation between binding and inhibition, we propose a three-stage binding-conformational change model of nucleotide regulation of uncoupling protein. We have now identified three nucleotides by site-directed mutagenesis that are essential for nucleotide interaction with uncoupling protein.
Modrianský
M
M
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97291-1000, USA.
Murdza-Inglis
D L
DL
Patel
H V
HV
Freeman
K B
KB
Garlid
K D
KD
D000818:Animals; D001120:Arginine; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D003062:Codon; D006153:Guanosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D007473:Ion Channels; D008239:Lysine; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D003062:Codon; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D000071256:Uncoupling Protein 1; D006153:Guanosine Diphosphate; D001120:Arginine; D008239:Lysine