Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9-Nup358 complex

Abstract

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 ubiquitination. Ub/Ubl conjugation occurs in sequential steps and requires the concerted action of E2 conjugating proteins and E3 ligases. 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 protein. Here we describe the 3.0-A 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.

Figure 1. Structure of SUMO-RanGAP1-Ubc9-Nup358/RanBP2 complex

A) Ribbon and transparent surface representation for the complex between SUMO-1 (yellow), Ubc9 (blue), RanGAP1 (pink), and Nup358/RanBP2 (magenta). Each protein is labeled. The SUMO C-terminal glycine (Gly97) and RanGAP1 Lys524 are represented by solid bonds located near the image center. B) Orthogonal view of the complex. C) Orthogonal view of the complex to highlight the extended Nup358/RanBP2 structure. N- and C- termini of Nup358/RanBP2 are denoted in italics. Structural graphics generated with PYMOL.

Figure 2. E2 active site in complex with RanGAP1-SUMO-1

Stereo view of the E2 active site in complex with SUMO-1-RanGAP1 in ribbon and solid bond representation. Residues are labeled and hydrogen bonding interactions indicated by dashed lines. SUMO-1, RanGAP1 and Ubc9 colored yellow, pink, and blue as in Figure 1.

Figure 3. Nup358/RanBP2 sequence alignment and E3-E2-SUMO-1 structure

A) IR1-M-IR2 elements. Secondary structure shown above the alignment. Single aa code colored for SUMO (yellow) and Ubc9 (blue) contacts. Nup358/RanBP2 IR constructs indicated by bars. Mutational analysis shown below IR1-M (‡ no defect; + impaired activity; − no activity). * above IR2 indicate identical amino acid positions between IR1 and IR2. B) Nup358/RanBP2 motifs I–V (magenta), SUMO-1 (yellow), and Ubc9 (blue). C) Nup358/RanBP2 motif I. D) Nup358/RanBP2 motif II. E) Nup358/RanBP2 motif II. F) Nup358/RanBP2 motif IV. G) Nup358/RanBP2 motif V. Residues labeled and hydrogen bonding interactions indicated by dashed lines.

Figure 4. Nup358/RanBP2 activities

Error bars are ±1 standard deviation. A) SUMO-1 RanGAP1 conjugation under multiple turnover plus (•) or minus (○) IR1* with gel insets. B) SUMO-1 p53 conjugation plus (•) or minus (○) IR1* with SUMO-1-RanGAP1. C) SUMO-1 conjugation rates for p53, IκB $\underset{¨}{\alpha }$ peptide. Nup358/RanBP2 constructs in E. D) Gel insets for (C) using p53. E) Rates (pM/sec) and relative rates for C. F) Gel insets for (G) using p53. G) Rates for p53, IκBα using Nup358/RanBP2 constructs or IR1-M-RanGAP1-Ubc9-SUMO-Nup358/RanBP2 (yellow). H) Rates (pM/sec) for G. I) Single turnover rates for p53 plus (red) or minus (black) IR1*.