2-STAGE NUCLEOTIDE-BINDING MECHANISM AND ITS IMPLICATIONS TO H-TRANSPORT INHIBITION OF THE UNCOUPLING PROTEIN FROM BROWN ADIPOSE-TISSUE MITOCHONDRIAL()
Sg. Huang et M. Klingenberg, 2-STAGE NUCLEOTIDE-BINDING MECHANISM AND ITS IMPLICATIONS TO H-TRANSPORT INHIBITION OF THE UNCOUPLING PROTEIN FROM BROWN ADIPOSE-TISSUE MITOCHONDRIAL(), Biochemistry, 35(24), 1996, pp. 7846-7854
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.