Structure of Importin13-Ubc9 complex: nuclear import and release of a key regulator of sumoylation

Abstract

Importin13 (Imp13) is an unusual β-karyopherin that is able to both import and export cargoes in and out of the nucleus. In the cytoplasm, Imp13 associates with different cargoes such as Mago-Y14 and Ubc9, and facilitates their import into the nucleus where RanGTP binding promotes the release of the cargo. In this study, we present the 2.8 Å resolution crystal structure of Imp13 in complex with the SUMO E2-conjugating enzyme, Ubc9. The structure shows an uncommon mode of cargo-karyopherin recognition with Ubc9 binding at the N-terminal portion of Imp13, occupying the entire RanGTP-binding site. Comparison of the Imp13-Ubc9 complex with Imp13-Mago-Y14 shows the remarkable plasticity of Imp13, whose conformation changes from a closed ring to an open superhelix when bound to the two different cargoes. The structure also shows that the binding mode is compatible with the sumoylated states of Ubc9. Indeed, we find that Imp13 is able to bind sumoylated Ubc9 in vitro and suppresses autosumoylation activity in the complex.

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

“…In the present study, [ 15 N- 1 H] heteronuclear single quantum correlation (HSQC) spectroscopy was used to determine which residues from Ubc9 are involved in the binding to CRMP2. In a HSQC spectrum, each amide residue yields a unique correlation peak, also called a chemical shift.…”

“…[ 15 N- 1 H] HSQC spectra of Ubc9, free and in complex with CRMP2 (1:2 Ubc9:CRMP2 ratio) were compared (Figure 2A). The NMR structure of Ubc9 has been solved and the chemical shift assignments are known (Biological Magnetic Resonance Bank (BMRB) ID 4132) [28].…”

“…A . Superposition of [ 15 N- 1 H] heteronuclear single quantum correlation spectroscopy (HSQC) spectra of [ 15 N]-labeled human Ubc9 (100 µM), free and in complex with unlabeled human CRMP2. CRMP2 was incubated with Ubc9 in a CRMP2:Ubc9 ratio of 2:1.…”

“…D . Average chemical shift changes for assigned residues of the [ 15 N]-labeled Ubc9 upon complex formation with CRMP2. The average chemical shift changes of cross-peaks are calculated as [(5Δδ HN ) 2 + (Δδ N ) 2 ] 1/2 ], where Δδ HN represents the chemical shift change of the amide proton, and Δδ N represents the chemical shift change of the amide nitrogen of an amino acid residue.…”

“…Based on this computational model, we predicted two major points of contact: Arginine 440 of CRMP2 to Glutamate 132 of Ubc9, and Valine 371 of CRMP2 to Alanine 131 of Ubc9. In this manuscript, we map the interaction of CRMP2 to Ubc9 using [ 15 N- 1 H] heteronuclear single quantum correlation spectroscopy (HSQC) and demonstrate that several residues that we predicted were indeed perturbed by CRMP2 binding. This result was largely recapitulated using the CRMP2 SUMO decoy, i.e.…”