Multitasking of the RanBP2 complex - Protein SUMOylation and export complex disassembly

Abstract

The essential, vertebrate-specific RanBP2/RanGAP1*SUMO1/Ubc9 complex is a fascinating macromolecular machine positioned at the nuclear pore complex during interphase. It is a unique composite SUMO E3 ligase and serves as a docking site for nuclear transport complexes. Moreover, the RanBP2 complex can stimulates Ran-GTP hydrolysis via its four Ran binding domains and the associated RanGAP activity. In the first part of this work, I wanted to obtain insight into the molecular mechanism of this composite E3 ligase. In order to unravel how it interacts with and activates its cognate E2 conjugating enzyme, I created a stable Ubc9∼SUMO thioester mimic that allowed in vitro interaction studies. I found evidence that SUMOylation via the RanBP2 complex depends on the formation of the catalytically productive, folded-back thioester conformation, similarly to ubiquitination. In collaboration with the group of Teresa Carlomagno (EMBL, Heidelberg), we mapped the interaction surfaces of the thioester mimic on the RanBP2 complex via NMR. Our analyses suggest that thioester-binding is not only achieved by the suspected interaction with RanBP2 itself, but also by a backside interaction between the SUMO1 molecule in the complex and the thioester-Ubc9. For this interaction, the complex opens up at its Ubc9/SUMO1-interface suggesting that it is not a static, but structurally dynamic entity during catalysis. In the second part of this work, I investigated possible roles of the RanBP2 complex for nuclear transport complexes, using a reconstituted version that contained RanGAP1, the SUMO E3 ligase region, two FG-repeat clusters and two Ran binding domains. I could show that the RanBP2 complex specifically binds and disassembles export complexes formed with the prototypic export receptor Crm1. The two FG-repeat clusters mediate RanBP2’s tight association with Crm1, which is followed by Ran binding domain-dependent cargo release and Ran-GTP hydrolysis. As the Crm1/RanBP2 interaction is compatible with RanBP2’s SUMO E3 ligase activity, my work also allows speculating about a possible Crm1-dependent substrate recruitment mechanism for the RanBP2 E3 ligase complex.