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. 2004 Oct 5;101(40):14373-8.
doi: 10.1073/pnas.0403498101. Epub 2004 Sep 23.

Identification of a SUMO-binding motif that recognizes SUMO-modified proteins

Jing Song  1 , Linda K Durrin, Thomas A Wilkinson, Theodore G Krontiris, Yuan Chen
Affiliations

Identification of a SUMO-binding motif that recognizes SUMO-modified proteins

Jing Song et al. Proc Natl Acad Sci U S A. .

Abstract

Posttranslational modification by the ubiquitin homologue, small ubiquitin-like modifier 1 (SUMO-1), has been established as an important regulatory mechanism. However, in most cases it is not clear how sumoylation regulates various cellular functions. Emerging evidence suggests that sumoylation may play a general role in regulating protein-protein interactions, as shown in RanBP2/Nup358 and RanGAP1 interaction. In this study, we have defined an amino acid sequence motif that binds SUMO. This motif, V/I-X-V/I-V/I, was identified by NMR spectroscopic characterization of interactions among SUMO-1 and peptides derived from proteins that are known to bind SUMO or sumoylated proteins. This motif binds all SUMO paralogues (SUMO-1-3). Using site-directed mutagenesis, we also show that this SUMO-binding motif in RanBP2/Nup358 is responsible for the interaction between RanBP2/Nup358 and sumoylated RanGAP1. The SUMO-binding motif exists in nearly all proteins known to be involved in SUMO-dependent processes, suggesting its general role in sumoylation-dependent cellular functions.

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Figures

Fig. 1.
Fig. 1.
Identification of SBM. (A) Superposition of the 1H-15N HSQC spectra of 15N/13C-labeled human SUMO-1, free (black) and in complex (red) with PIASX-P. The sequence of PIASX-P is shown above the spectra. (B) Superposition of the TOCSY spectrum (black) of free PIASX-P and the 15N-filtered TOCSY spectrum (red) of PIASX-P in complex of 1:1 molar ratio with 15N/13C-labeled SUMO-1. Resonance assignments of the free peptide are indicated. (C) Superposition of the 1H-15N HSQC spectra of SUMO-1, free (black) and in complex with PIASX-N (red). The residues in SUMO-1 that are significantly affected by the complex formation are indicated with their assignments. (D-F) ITC measurements for the interaction between PIASX-P or PIASX-N and SUMO-1 or -3. Experimental details are provided in Materials and Methods. (G) Summary of thermodynamic parameters obtained from the ITC measurements shown in D-F.
Fig. 2.
Fig. 2.
Interaction between SUMO-1 and PML. (A) Superposition of the 1H-15N HSQC spectra of SUMO-1, free (black) and in complex with PML-P (red). The sequence of PML-P is shown above the spectra. The residues in SUMO-1 that are significantly affected by the complex formation are indicated with their assignments. (B) Superposition of the TOCSY spectrum of free PML-P (black) and the 15N-filtered TOCSY spectrum of PML-P in complex with 15N-13C-labeled SUMO-1 (red). Resonance assignment of the free peptide is indicated.
Fig. 3.
Fig. 3.
Identification of the consensus sequence. (A-C) Plots of chemical shift changes of SUMO-1 in complex with PIASX-P (A), PML-P (B), and SAE2-P (C) vs. residue number. Chemical shift changes were calculated as square root of (25 × Δδ1H2 + Δδ15N2) to compensate for the difference in chemical shift dispersion between 1H and 15N nuclei. (D) Sequence alignment of the four synthetic peptides (PIASX-P, PIASX-N, PML-P, and SAE2-P) used in the studies to define the sequence requirement for binding SUMO. Sequences are aligned to reveal the conserved motif V-X-V/I-V/I.
Fig. 4.
Fig. 4.
The binding site on SUMO-1 for the SBM. (A) Ribbon diagram of the 3D structure of SUMO-1. Residues that show significant chemical shift perturbation upon binding to the peptides are indicated in red, as suggested by the results shown in Fig. 3. (B) Surface representation of the 3D structure of SUMO-1. The orientation of the molecule in B is the same as that in A. The color spectrum of red to blue corresponds to changes from negative to positive potentials over a range of -5to +5 KB/electron. Surface hydrophobic residues are indicated in green.
Fig. 5.
Fig. 5.
Functional implications of the SBM in RanBP2/Nup358. (A) Design of the mutations within residues 2596-2836 of RanBP2/Nup358. (B) Full length RanGAP1 was translated in vitro by using rabbit reticulocyte transcription/translation extracts in the presence of [35S]methionine. Both RanGAP1 and sumoylated RanGAP1 were produced during the translation process, as shown in the first lane. The asterisks indicate truncated versions of RanGAP1. Translated proteins were assayed for binding to the wild-type or mutant RanBP2/Nup358 fragments, as described in Materials and Methods. (C) Pull down of Ubc9 by the wild-type and mutant RanBP2/Nup358 fragment, as described in Materials and Methods. Western blot with anti-His-tag antibody was used to detect Ubc9.
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