Whitby F G, Xia G, Pickart C M, Hill C P
Department of Biochemistry, University of Utah, Salt Lake City, Utah 84132, USA.
J Biol Chem. 1998 Dec 25;273(52):34983-91. doi: 10.1074/jbc.273.52.34983.
The NEDD8/Rub1 class of ubiquitin-like proteins has been implicated in progression of the cell cycle from G1 into S phase. These molecules undergo a metabolism that parallels that of ubiquitin and involves specific interactions with many different proteins. We report here the crystal structure of recombinant human NEDD8 refined at 1.6-A resolution to an R factor of 21.9%. As expected from the high sequence similarity (57% identical), the NEDD8 structure closely resembles that reported previously for ubiquitin. We also show that recombinant human NEDD8 protein is activated, albeit inefficiently, by the ubiquitin-activating (E1) enzyme and that NEDD8 can be transferred from E1 to the ubiquitin conjugating enzyme E2-25K. E2-25K adds NEDD8 to a polyubiquitin chain with an efficiency similar to that of ubiquitin. A chimeric tetramer composed of three ubiquitins and one histidine-tagged NEDD8 binds to the 26 S proteasome with an affinity similar to that of tetraubiquitin. Seven residues that differ from the corresponding residues in ubiquitin, but are conserved between NEDD8 orthologs, are candidates for mediating interactions with NEDD8-specific partners. One such residue, Ala-72 (Arg in ubiquitin), is shown to perform a key role in selecting against reaction with the ubiquitin E1 enzyme, thereby acting to prevent the inappropriate diversion of NEDD8 into ubiquitin-specific pathways.
类泛素蛋白NEDD8/Rub1与细胞周期从G1期进入S期的进程有关。这些分子经历的代谢过程与泛素相似，并且涉及与许多不同蛋白质的特异性相互作用。我们在此报告重组人NEDD8的晶体结构，其在1.6埃分辨率下精修，R因子为21.9%。正如从高序列相似性（57%相同）所预期的那样，NEDD8结构与先前报道的泛素结构非常相似。我们还表明，重组人NEDD8蛋白可被泛素激活酶（E1）激活，尽管效率不高，并且NEDD8可以从E1转移到泛素缀合酶E2-25K。E2-25K将NEDD8添加到多聚泛素链上的效率与泛素相似。由三个泛素和一个组氨酸标记的NEDD8组成的嵌合四聚体与26S蛋白酶体结合的亲和力与四聚泛素相似。与泛素中相应残基不同但在NEDD8直系同源物之间保守的七个残基，是介导与NEDD8特异性伴侣相互作用的候选者。其中一个这样的残基，Ala-72（泛素中的Arg），在选择不与泛素E1酶反应中起关键作用，从而防止NEDD8不适当地转向泛素特异性途径。
