Labeling of two different regions of the nucleotide binding site of the uncoupling protein from brown adipose tissue mitochondria with two ATP-analogs

Abstract

The nucleotide binding site of the uncoupling protein (UCP) from brown adipose tissue was mapped by photoaffinity labeling with 2-azidoadenosine 5'-triphosphate (2-azido-ATP) and by affinity labeling with 3'-O-(5-fluoro-2,4-dinitrophenyl)adenosine 5'-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 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)

EPR spectroscopy of 5‐DOXYL‐stearic acid bound to the mitochondrial uncoupling protein reveals its competitive displacement by alkylsulfonates in the channel and allosteric displacement by ATP

“…Purine nucleotide inhibition is caused by conformational changes which make the anion binding site inacessible for anions. The conformational changes originate in and progress from the water-filled cavity of the ATP binding site [23], where ATP may reach the 'bottom' of the protein.…”

Structure–Function Relationship in UCP1

“…14 The nucleotide binding site had been mapped by photoaf®nity labelling with 2 and 8 azido ATP, as well as with 3--¯uoronitrophenyl ATP. 15 Three residues were identi®ed in the covalent attachment, located in the matrix loop of the third domain. Accessibility from the cytosolic site by ATP derivatives to the matrix loop led to the suggestion that these loops protrude into the membrane region surrounded by the helices.…”

Important amino acid residues of potato plant uncoupling protein (StUCP)

“…When Arg 276 was either substituted in a mutated UCP1 protein (34) or modified by phenylglyoxal and 2,3-butadione (32), purine nucleotide binding and gating were absent. Since the proximal third matrix segment was photolabeled at three different positions with 8-azido-, 2-azido-and 3-O-(5-fluoro-2,4-dinitrophenyl) adenosine 5-triphosphate (FNDP-ATP) (35), and since the deletion of residues 261-269 resulted in the lack of nucleotide inhibition (36), it was concluded that the main location of the nucleotide-binding site in UCP1 was located between the fifth and sixth transmembrane segments. This site probably forms a waterfilled cavity which penetrates deeply into the membrane close to the opposite surface (35).…”