Labeling of Two Different Regions of the Nucleotide Binding Site of the Uncoupling Protein from Brown Adipose Tissue Mitochondria with Two ATP Analogs

Peter Mayinger, Martin Klingenberg

Research output: Contribution to journalArticle

46 Scopus citations

Abstract

The nucleotide binding site of the uncoupling protein (UCP) from brown adipose tissue was mapped by photoaffinity labeling with 2-azidoadenosine 5ʹ-triphosphate (2-azido-ATP) and by affinity labeling with 3ʹ-0-(5-fluoro-2, 4-dinitrophenyl)adenosine 5ʹ-triphosphate (FDNP-ATP). Both analogs bind with high affinity and specificity to the UCP in intact mitochondria, as well as to the isolated solubilized protein. Reversible binding at 4 °C in the dark is competitively blocked by GTP. Like the natural ligands ATP and GTP, both analogs are capable of inhibiting the H+/OH- conductance of the UCP as measured in proteoliposomes with reconstituted UCP. 2-azido-ATP was incorporated into UCP in mitochondria in the presence of carboxyatractylate, while FDNP-ATP was inserted into isolated UCP by prolonged incubation at room temperature under pH variation. Both reactions can be blocked by GTP. The incorporation of 2-azido-ATP could be localized between residues 258 and 283 by cleavage with CNBr. Solid-phase sequencing of the homoserine-linked radioactive peptide indicated that the 2-azido-ATP was linked to threonine-263. The incorporation of FDNP-ATP could be assigned by cleavage with CNBr and alternatively with trypsin at a locus of covalent attachment between residues 238 and 255. On the basis of published data that no tyrosine participates in nucleotide binding of the UCP, the probable residue reacting with FDNP-ATP is cysteine-253. According to these results and the recently published experiments with 8-azido-ATP, the third hydrophilic domain of the triple-domain primary structure forms the nucleotide binding site between the two putative membrane helices α-5 and α-6. Within this region, the ATP seems to interact at the 2-adenine position with threonine-263 and at the 3ʹ-OH group of the ribose with cysteine-253.

Original languageEnglish (US)
Pages (from-to)10536-10543
Number of pages8
JournalBiochemistry
Volume31
Issue number43
DOIs
StatePublished - Feb 1 1992

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Labeling of Two Different Regions of the Nucleotide Binding Site of the Uncoupling Protein from Brown Adipose Tissue Mitochondria with Two ATP Analogs'. Together they form a unique fingerprint.

  • Cite this