Huang S G, Klingenberg M
Institute of Physical Biochemistry, University of Munich, Germany.
Biochemistry. 1996 Jun 18;35(24):7846-54. doi: 10.1021/bi960244p.
The uncoupling protein (UCP) from brown adipose tissue mitochondria is the simplest H+ translocator known. H+ transport is regulated by fatty acids as activators and by pruine nucleotides as inhibitors. Nucleotide binding again is strongly influenced by the pH [Klingenberg, M. (1988) Biochemistry 27, 781-791]. Previously, by using fluorescent 2'-O-dansyl (DANS) derivatives of purine nucleotides, a two-stage binding mechanism was unraveled with a slow transition from a loose into a tight conformational state in the isolated UCP [Huang, S.-G., & Klingenberg, M. (1995) Biochemistry 34, 349-360]. Whereas with the unsubstituted nucleotides the transition to the tight state is nearly complete, various DANS and DAN (dimethylaminonaphthoyl) nucleotides bind more to the loose state. Here we investigated the relationships between the two-stage nucleotide binding and the inhibition of the H+ transport activity in reconstituted proteoliposomes. Further, limited tryptic digestion was used as an indicator of conformational change induced by the nucleotide binding in the isolated protein. The inhibition of H+ transport activity in reconstituted UCP proteoliposomes correlated only with the fraction of tight state of nucleotide binding. Unsubstituted nucleotides (ATP, GTP, and ADP) as well as DANSGTP inhibit fully the H+ transport, whereas DANSATP and DANSADP inhibit only to about 50%, and DANSAMP is nearly ineffective. Even for the loose conformational state the nucleotide derivatives exhibit considerable affinity. This allows DANSAMP to replace prebound ATP from UCP and relieve the inhibition of H+ transport by reversing the distribution of UCP from the tight into the loose conformational state. The pH dependence of the fraction of nucleotide binding in the tight state correlates closely with the pH dependence of the degree of H+ transport inhibition. Titration with DANS nucleotides of UCP incorporated into phospholipid vesicles revealed that over 70% of binding sites had an affinity comparable with that for the isolated UCP while the remaining sites displayed substantially lower affinity, due to nonhomogeneity of the reconstituted system. The sensitivity against trypsin digestion is inversely correlated with the fraction of nucleotide binding in the tight state. Whereas unsubstituted nucleotides and DANSGTP protect strongly against trypsinolysis, DANSATP and DANSADP do only partially, and DANSAMP does not at all. The counteracting influences of the DANS substitution are shown with DANSAMP, which has an affinity comparable to that of DANSATP or DANSADP but cannot form the tight inhibited complex. These data show that nucleotide binding only in the tight state is associated with a strong conformational change, which further causes an inhibition of H+ transport. In conclusion, 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 remains to variable degrees in the loose state with DANS and DAN derivatives. The DANS group hinders progressively the transition to the tight state as the binding affinity of the underlying nucleotide decreases.
棕色脂肪组织线粒体中的解偶联蛋白（UCP）是已知最简单的H⁺转运体。H⁺转运受脂肪酸作为激活剂以及嘌呤核苷酸作为抑制剂的调节。核苷酸结合同样受到pH的强烈影响[克林根贝格，M.（1988年）《生物化学》27卷，781 - 791页]。此前，通过使用嘌呤核苷酸的荧光2'-O-丹磺酰（DANS）衍生物，在分离的UCP中揭示了一种两阶段结合机制，即从松散构象状态缓慢转变为紧密构象状态[黄，S.-G.，& 克林根贝格，M.（1995年）《生物化学》34卷，349 - 360页]。对于未取代的核苷酸，向紧密状态的转变几乎是完全的，而各种DANS和DAN（二甲基氨基萘甲酰）核苷酸更多地结合到松散状态。在此，我们研究了重组蛋白脂质体中两阶段核苷酸结合与H⁺转运活性抑制之间的关系。此外，有限的胰蛋白酶消化被用作分离蛋白中核苷酸结合诱导的构象变化的指标。重组UCP蛋白脂质体中H⁺转运活性的抑制仅与核苷酸结合紧密状态的比例相关。未取代的核苷酸（ATP、GTP和ADP）以及DANSGTP完全抑制H⁺转运，而DANSATP和DANSADP仅抑制约50%，DANSAMP几乎无效。即使对于松散构象状态，核苷酸衍生物也表现出相当大的亲和力。这使得DANSAMP能够从UCP取代预先结合的ATP，并通过将UCP的分布从紧密构象状态转变为松散构象状态来解除对H⁺转运的抑制。紧密状态下核苷酸结合比例的pH依赖性与H⁺转运抑制程度的pH依赖性密切相关。用DANS核苷酸滴定掺入磷脂囊泡中的UCP表明，超过70%的结合位点具有与分离的UCP相当的亲和力，而其余位点由于重组系统的不均匀性显示出显著较低的亲和力。对胰蛋白酶消化的敏感性与紧密状态下核苷酸结合的比例呈负相关。未取代的核苷酸和DANSGTP对胰蛋白酶水解有很强的保护作用，DANSATP和DANSADP仅提供部分保护，而DANSAMP则完全没有保护作用。DANSAMP显示出DANS取代的抵消作用，其亲和力与DANSATP或DANSADP相当，但不能形成紧密的抑制复合物。这些数据表明，仅紧密状态下的核苷酸结合与强烈的构象变化相关，这进一步导致H⁺转运的抑制。总之，UCP可以存在于松散的非抑制性构象状态和紧密的抑制性构象状态。这两种构象之间的平衡在未取代的核苷酸作用下向紧密状态移动，但在DANS和DAN衍生物作用下在松散状态下保持不同程度。随着基础核苷酸的结合亲和力降低，DANS基团逐渐阻碍向紧密状态的转变。
