2-STAGE NUCLEOTIDE-BINDING MECHANISM AND ITS IMPLICATIONS TO H-TRANSPORT INHIBITION OF THE UNCOUPLING PROTEIN FROM BROWN ADIPOSE-TISSUE MITOCHONDRIAL()

The uncoupling protein (UCP) from brown adipose tissue mitochondria is the simplest H+ translocator known. H+ transport is regulated by fatt y acids as activators and by purine nucleotides as inhibitors. Nucleot ide binding again is strongly influenced by the pH [Klingenberg, M. (1 988) Biochemistry 27, 781-791]. Previously, by using fluorescent 2'-O- dansyl (DANS) derivatives of purine nucleotides, a two-stage binding m echanism was unraveled with a slow transition from a loose into a tigh t conformational state in the isolated UCP [Huang, S.-G., & Klingenber g, M. (1995) Biochemistry 34, 349-360]. Whereas with the unsubstituted nucleotides the transition to the tight state is nearly complete, var ious DANS and DAN (dimethylaminonaphthoyl) nucleotides bind more to th e loose state. Here we investigated the relationships between the two- stage nucleotide binding and the inhibition of the H+ transport activi ty in reconstituted proteoliposomes. Further, limited tryptic digestio n was used as an indicator of conformational change induced by the nuc leotide binding in the isolated protein. The inhibition of H+ transpor t activity in reconstituted UCP proteoliposomes correlated only with t he fraction of tight state of nucleotide binding. Unsubstituted nucleo tides (ATP, GTP, and ADP) as well as DANSGTP inhibit fully the Hf tran sport, whereas DANSATP and DANSADP inhibit only to about 50%, and DANS AMP is nearly ineffective. Even for the loose conformational state the nucleotide derivatives exhibit considerable affinity. This allows DAN SAMP to replace prebound ATP from UCP and relieve the inhibition of H transport by reversing the distribution of UCP from the tight into th e loose conformational state. The pH dependence of the fraction of nuc leotide binding in the tight state correlates closely with the pH depe ndence of the degree of H+ transport inhibition. Titration with DANS n ucleotides of UCP incorporated into phospholipid vesicles revealed tha t over 70% of binding sites had an affinity comparable with that for t he isolated UCP while the remaining sites displayed substantially lowe r affinity, due to nonhomogeneity of the reconstituted system. The sen sitivity against trypsin digestion is inversely correlated with the fr action of nucleotide binding in the tight state. Whereas unsubstituted nucleotides and DANSGTP protect strongly against trypsinolysis, DANSA TP and DANSADP do only partially, and DANSAMP does not at all. The cou nteracting influences of the DANS substitution are shown with DANSAMP, which has an affinity comparable to that of DANSATP or DANSADP but ca nnot form the tight inhibited complex. These data show that nucleotide binding only in the tight state is associated with a strong conformat ional change, which further causes an inhibition of H+ transport. In c onclusion, UCP can exist in a loose noninhibited and a tight inhibited conformational state. The equilibrium between these two conformations is shifted to the tight state with unsubstituted nucleotides but rema ins to variable degrees in the loose state with DANS and DAN derivativ es. The DANS group hinders progressively the transition to the tight s tate as the binding affinity of the underlying nucleotide decreases.