SUMO-1 (for small ubiquitin-related modifier) belongs to the ubiquitin (Ub) and ubiquitin-like (Ubl) protein family. SUMO conjugation occurs on specific lysine residues within protein targets, regulating pathways involved in differentiation, apoptosis, the cell cycle and responses to stress by altering protein function through changes in activity or cellular localization or by protecting substrates from ubiquitination1,2. Ub/Ubl conjugation occurs in sequential steps and requires the concerted action of E2 conjugating proteins and E3 ligases1,2. In addition to being a SUMO E3, the nucleoporin Nup358/RanBP2 localizes SUMO-conjugated RanGAP1 to the cytoplasmic face of the nuclear pore complex by means of interactions in a complex that also includes Ubc9, the SUMO E2 conjugating protein3,4,5,6. Here we describe the 3.0-Å crystal structure of a four-protein complex of Ubc9, a Nup358/RanBP2 E3 ligase domain (IR1-M) and SUMO-1 conjugated to the carboxy-terminal domain of RanGAP1. Structural insights, combined with biochemical and kinetic data obtained with additional substrates, support a model in which Nup358/RanBP2 acts as an E3 by binding both SUMO and Ubc9 to position the SUMO–E2-thioester in an optimal orientation to enhance conjugation.
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We thank M. J. Matunis for the original clone containing Nup358/RanBP2 (residues 2,596–2,836), and K. R. Rajashankar and A. Yunus for discussion and for reagents that contributed to this work. Use of the Advanced Photon Source (APS) is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences. Use of the SGX Collaborative Access Team beamline facilities at Sector 31 of the APS was provided by Structural GenomiX, Inc., which constructed and operates the facility. D.R. and C.D.L. were supported in part by a National Institutes of Health grant. C.D.L. acknowledges support from the Rita Allen Foundation.
Coordinates have been deposited with the Protein Data Bank under accession number 1Z5S. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
Crystallographic data, phasing, and refinement statistics. (DOC 53 kb)
Activities of the Nup358/RanBP2 E3 with RanGAP1 and RanGAP1 (F562A). a) Activity under multiple turnover conditions with RanGAP1 and RanGAP1(F562A). b) Activity under single turnover conditions with RanGAP1 and RanGAP1(F562A). (PDF 61 kb)
Biochemical activities of Nup358/RanBP2 with SUMO-2 and SUMO-3. a) For SUMO-2 conjugation. b) For SUMO-3 conjugation. c) Table of rate constants and relative rates for reactions in a and b. (PDF 29 kb)
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Reverter, D., Lima, C. Insights into E3 ligase activity revealed by a SUMO–RanGAP1–Ubc9–Nup358 complex. Nature 435, 687–692 (2005). https://doi.org/10.1038/nature03588
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