br Concluding Remarks Recent studies have provided
Concluding Remarks Recent studies have provided unprecedented details of APC/C structure and enzymology, which explain how the activity of this massive E3 ligase is controlled, and how ubiquitylation is achieved to temporally regulate cell division. Although one pervasive question has been why the APC/C has such an enormous molecular mass, it seems that the large size enables both nuanced and extreme conformational changes – and their coupling to phosphorylation and the binding of many partner proteins. Step-by-step regulation is achieved through each complex allowing precisely the needed APC/C functions while excluding others (Figure 4). For example, apo, unphosphorylated APC/C excludes CDC20 and UBE2C, while phosphorylated APC/C allows coactivator binding. The many activities of APC/CCDC20 are further tuned, including MCC in a closed and inactive state; an open configuration that excludes APC/C substrates but allows UBE2C-catalyzed ubiquitylation of MCC, and substrate bound in a manner that excludes MCC and allows UBE2C for its direct modification. Ubiquitylated substrates and UBE2S capture yet alternative locations on the cullin–RING core for polyubiquitylation to drive progression through mitosis, while EMI1 subsequently prevents substrate and E2 binding to allow MJ33 lithium salt accumulation and another cell cycle. Despite the wealth of structural data, many open questions remain (see Outstanding Questions), especially relating to how these discrete APC/C complexes transition from one state – or binding partner and activity – to another. It seems likely that the multisite, avid nature of the interactions of APC/C could contribute to this regulation, as all elements within most APC/C partners are required for their high-affinity binding. It seems likely that the dismantling of one interaction, for example through a post-translational modification or the binding of another protein, could precipitate disassembly through a domino-like effect. Future studies will also be required to visualize emerging APC/C regulation, including by phosphorylation, SUMOylation, association with enigmatic binding partners 91, 92, 93, 94, and localization of APC/C to its different regulators and substrates within cells. In addition to APC/C, humans are estimated to express >500 different E3 ligases, roughly half of which are CRLs whose catalytic core becomes mobile upon activation . However, because few activity states have been structurally visualized for most E3 ligases, the studies on APC/C described herein provide paradigmatic molecular principles determining distinct E3 ligase activities across a biological pathway. Although the structural details will differ between ligases, it seems that much like APC/C, E3s are generally restrained in inactive conformations until needed, conformationally flexible when activated, and harnessed into distinct conformations for different functions.
Acknowledgments We thank our colleagues who have contributed to our understanding of the cell cycle and the structure and function of APC/C, and apologize to those whose work we were unable to cite here. We thank J. Rajan Prabu for making the movies showing APC/C conformational changes. Our work on APC/C is funded by NIHR35GM128855 and UCRF (NGB), Boehringer Ingelheim, Austrian Science Fund, Austrian Research Promotion Agency, and the European Community (JMP); the Max Planck Society (HS and BAS); and ALSAC, NIH R37GM065930 and P30CA021765 (BAS).
Memoir Some of the earliest work on the function of the tRNA initiating protein translation, the N-Formyl-Methionyl-tRNA, was described by Brian FC Clark , , , , , and a follow up study led to the crystal structure of yeast phenylalanine tRNA in the laboratory headed by Aaron Klug . Brian Clark was initially head-hunted to join him at Aarhus University and lead the Department for Biostructural Chemistry, which was the perfect setting for him to continue his vision towards understanding the fundamentals of the genetic code. As a university teacher, Brian was an inspirational speaker and excellent at conveying the message. His teaching always included keeping an open mind and only by combining all the present data, both the structural and the functional, a conclusion could be drawn – an approach that still is integrated into the Biostructural Department today.