(15) pH REGULATION OF NUCLEOTIDE BINDING TO UNCOUPLING PROTEIN (UCP-1)

Karim S. Echtay*, Martin Bienengraeber and Martin Klingenberg
Institute of Physical Biochemistry, University of Munich, Schillerstrsse 44, 80336 Munich, Germany

Nucleotide binding controls the activity of the uncoupling protein (UCP-1 ) of brown adipose tissue. The nucleotide binding to UCP-1 is strongly pH dependent. It has been proposed that protonisation of a carboxylic group (pK 4.5) and of a histidine (pK 7.2) regulate access to the binding pocket of the phosphate moiety. We identify here the carboxylic group as E190 for the pH control of both nucleoside di- (NDP) and triphosphate (NTP) and the histidine as H214 for NTP only. These two pH sensors are evaluated using site-directed mutagenesis of UCP-1 expressed in S.cerevisiae. The expressed protein was purified and reconstituted into liposomes for studying H+ and Cl- transport. We studied NDP and NTP binding to isolated protein by determining the dissociation constant (KD) and the kinetic of binding at different pH. The results show that E190 and H214 are not involved in H+ and Cl- transport, but that their mutation drastically decreases pH dependency of nucleotide binding, E190Q for both of NDP and NTP binding and H214N and H214W only of NTP binding. The KD for GTP binding to wt increased 20-fold from 0.39 µM at pH 6.0 to 7.9 µM at pH 7.5, while in the mutants this increase was only 2.5 in E190Q, 6 in H214N and 1.6-fold in H214W. The binding rate (kon) of GTP decreased 10-fold from pH 6.0 to 7.5 in wt and only 4-fold in the mutants. For ADP binding, the pH sensitivity was the same for wt and H214 mutants but almost lost for E190Q. These results support the proposal that E190 forms an ion pair at the entrance to the phosphate moiety binding cleft. On protonisation of E190, the binding site opens and the positive residue becomes available for the phosphate moiety binding of NDP and NTP. H214 plays a regulatory role only for NTP binding. In the neutral form H214 protrudes into the binding pocket, thus interfering with insertion of NTP ?-phosphate. On protonation, HisH+ is retracted by a background -CO2-, thus enlarging the pocket to accommodate the ?-phosphate. H214 is not involved in ADP binding.

[1] Klingenberg M. (1988) Biochemistry 27:781-791.
[2] Echtay KS, Bienengraeber M, Klingenberg M (1997) Biochemistry 36:8253-8260
[3] Echtay KS, Bienengraeber M, Winkler E, Klingenberg M. (1998) J. Biol. Chem. (in press)