Insights into High Affinity Small Ubiquitin-like Modifier (SUMO) Recognition by SUMO-interacting Motifs (SIMs) Revealed by a Combination of NMR and Peptide Array Analysis

Andrew T Namanja,Yi-Jia Li,Yang Su,Steven Wong,Jingjun Lu,Loren T Colson,Chenggang Wu,Shawn S.C Li,Yuan Chen

doi:10.1074/jbc.m111.293118

Andrew T Namanja, Yi-Jia Li + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m111.293118

Copy DOI

Abstract

The small ubiquitin-like modifiers (SUMOs) regulate many essential cellular functions. Only one type of SUMO-interacting motif (SIM) has been identified that can extend the β-sheet of SUMO as either a parallel or an antiparallel strand. The molecular determinants of the bound orientation and paralogue specificity of a SIM are unclear. To address this question, we have conducted structural studies of SUMO1 in complex with a SUMO1-specific SIM that binds to SUMO1 with high affinity without post-translational modifications using nuclear magnetic resonance methods. In addition, the SIM sequence requirements have been investigated by peptide arrays in comparison with another high affinity SIM that binds in the opposing orientation. We found that antiparallel binding SIMs tolerate more diverse sequences, whereas the parallel binding SIMs prefer the more strict sequences consisting of (I/V)DLT that have a preference in high affinity SUMO2 and -3 binding. Comparison of two high affinity SUMO1-binding SIMs that bind in opposing orientations has revealed common SUMO1-specific interactions needed for high affinity binding. This study has significantly advanced our understanding of the molecular determinants underlining SUMO-SIM recognition.

Highlights

The small ubiquitin-like modifiers (SUMOs)-interacting motif (SIM) mediates SUMO-dependent regulation
Structure of Highly Specific SUMO1-binding SUMO-interacting motif (SIM) in Complex with SUMO1—As discussed in the Introduction, the M-IR2 domain of RanBP2 harbors a high affinity SIM that is specific to SUMO1 in the absence of a post-translational modification
That the antiparallel M-IR2 SIM tolerates more diverse amino acid sequences than the parallel PIASX SIM indicates that backbone hydrogen bonds, as well as the ␤-strand propensity of the SIM residues [27], make more significant contributions than specific side chain interactions to formation of the complex

Summary

Background

The SUMO-interacting motif (SIM) mediates SUMO-dependent regulation. Results: The structure of a SUMO1-specific SIM in complex with SUMO1 is solved, and the SIM sequence requirements are identified by peptide arrays. The molecular determinants of the bound orientation and paralogue specificity of a SIM are unclear To address this question, we have conducted structural studies of SUMO1 in complex with a SUMO1-specific SIM that binds to SUMO1 with high affinity without post-translational modifications using nuclear magnetic resonance methods. SIM Sequence Requirements for High Affinity SUMO Binding [15], only one type of SIM is prevalent in SUMO-mediated protein-protein interactions of all SUMO paralogues [14, 16]. To improve our understanding of the sequence requirements of SIMs for high affinity and high specificity interactions, we carried out NMR analysis of the structure of SUMO1 in complex with the high affinity SUMO1-specific M-IR2 SIM We found that this SIM binds SUMO1 as an antiparallel ␤-strand. The molecular insights obtained here provide further understanding of the sequence requirements and mechanism underlying SUMO-SIM recognition

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION