Identification of the pH Sensor for Nucleotide Binding in the Uncoupling Protein from Brown Adipose Tissue

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Abstract
The transport inhibiting nucleotide binding to the uncoupling protein (UCP) has a unique pH dependence and has been postulated to be controlled by the dissociation state of a carboxyl group in UCP with pK 4.5 and, in addition only for the nucleoside triphosphate, by a group with pK 7.2. To prove this assumption and to identify the carboxyl group, Woodward reagent K (WRK) was applied to UCP. In mitochondria, WRK was found to inhibit binding of GTP in a noncompetitive manner using WRK in the millimolar range. In isolated UCP, GTP binding is inhibited by WRK at a 1 to 2 ratio to UCP, suggesting that WRK primarily reacts with only one carboxyl group. Prebound GTP protects against WRK reaction as monitored by GTP binding. The protection decreases from pH 5 to 7 due to better reactivity of WRK and less tight GTP binding. WRK does not inhibit H+ transport by UCP but prevents GTP inhibition of H+ transport. For elucidating the WRK target residue, the WRK derivatized group was labeled with [3H] by reduction with [3H]NaBH4. Both GTP and GDP largely protected against WRK-dependent [3H] labeling. CNBr fragmentation identified the region T121−M197 as the [3H] incorporation site. Combined CNBr and tryptophane cleavage by the reagent 3-bromo-3-methyl-2-((2-nitrophenyl)thio)-3H-indole (BNPS) allowed to further delimit the 2.8 kDa peptide W173−M197 as the [3H] label carrier which contains two acid residues E190 and D195. To further identify the residue, limited tryptic digestion in sarcosyl-treated UCP was performed, and a tryptic fragment enclosing E190 and D195 was isolated which carried most of the [3H] label. Edman degradation showed the major [3H] label at the eighth position corresponding to E190 and no peak at D195. Thus, the original postulate of the pH-sensing carboxyl group regulating both the nucleoside di- and triphosphate binding has been verified. It is identified as E190 situated in the fourth transmembrane helix. In total, now four residues close to the nucleotide binding sites in UCP have been determined.