Structural Basis for E2-Mediated SUMO Conjugation Revealed by a Complex between Ubiquitin-Conjugating Enzyme Ubc9 and RanGAP1

Abstract

E2 enzymes catalyze attachment of ubiquitin and ubiquitin-like proteins to lysine residues directly or through E3-mediated reactions. The small ubiquitin-like modifier SUMO regulates nuclear transport, stress response, and signal transduction in eukaryotes and is essential for cell-cycle progression in yeast. In contrast to most ubiquitin conjugation, the SUMO E2 enzyme Ubc9 is sufficient for substrate recognition and lysine modification of known SUMO targets. Crystallographic analysis of a complex between mammalian Ubc9 and a C-terminal domain of RanGAP1 at 2.5 A reveals structural determinants for recognition of consensus SUMO modification sequences found within SUMO-conjugated proteins. Structure-based mutagenesis and biochemical analysis of Ubc9 and RanGAP1 reveal distinct motifs required for substrate binding and SUMO modification of p53, IkappaBalpha, and RanGAP1.

A framework for exhaustively mapping functional missense variants

“…Indeed, a comparison with co‐crystal structure data (Gareau et al , 2012) shows that many of the hypercomplementing residues are on the surface proximal to the substrate, with some directly contacting the substrate's sumoylation motif (Fig 3C). In vitro sumoylation assays performed previously for a small number of UBE2I mutants revealed increased sumoylation for some substrates (Bernier‐Villamor et al , 2002). Comparing our map with these sumoylation assay results, we saw that cases of hypercomplementation were enriched for substrate specificity shift (Appendix Fig S6C).…”

“…Images from in vitro sumoylation assays performed for UBE2I variants by (Bernier‐Villamor et al , 2002) were scored by visual inspection while blinded to the underlying variant information. Scores were then represented as a heatmap and compared complementation scores from the UBE2I map.…”

“…All final variant maps and associated data tables can be found in Dataset EV1 and can be downloaded at http://dalai.mshri.on.ca/~jweile/projects/dmsData/ or from the Biostudies database (www.ebi.ac.uk/biostudies/), accession number S‐BSST60. Original data for the in vitro sumoylation analysis can be found in Bernier‐Villamor et al (2002). …”

Chemical shift perturbation mapping of the Ubc9-CRMP2 interface identifies a pocket in CRMP2 amenable for allosteric modulation of Nav1.7 channels

“…Reportedly, residues Asn121 to Ala131 directly interact with the SUMOylation motif of target proteins while residues Glu132-Arg141 have been shown to be important for selective target recognition [13], [18], [22]. More specifically, mutations of Ubc9 residues Gln126, Gln130, Ala131, Glu132, Tyr134, and Thr135 led to a reduction in SUMOylation of protein targets [16].…”

“…Within target protein(s), Ubc9 recognizes a SUMOylation motif – “Ψ-K-x-D/E” – where Ψ represents a hydrophobic residue and K is the SUMO acceptor lysine [17]. In addition to this consensus sequence, some SUMO substrate proteins, like the mammalian guanosine triphosphate (GTP)ase-activating protein (RanGAP1), exhibit a second contact surface with Ubc9, which is believed to promote higher SUMOylation efficiency [18]. We generated a computational model of the CRMP2-Ubc9-SUMO interaction [11].…”