A conserved and buried edge-to-face aromatic interaction in small ubiquitin-like modifier (SUMO) has a role in SUMO stability and function

Kiran Sankar Chatterjee,Vasvi Tripathi,Ranabir Das

doi:10.1074/jbc.ra118.006642

Abstract

Aromatic amino acids buried at a protein's core are often involved in mutual paired interactions. Ab initio energy calculations have highlighted that the conformational orientations and the effects of substitutions are important for stable aromatic interactions among aromatic rings, but studies in the context of a protein's fold and function are elusive. Small ubiquitin-like modifier (SUMO) is a common post-translational modifier that affects diverse cellular processes. Here, we report that a highly conserved aromatic triad of three amino acids, Phe36-Tyr51-Phe64, is a unique SUMO signature that is absent in other ubiquitin-like homologous folds. We found that a specific edge-to-face conformation between the Tyr51-Phe64 pair of interacting aromatics is vital to the fold and stability of SUMO. Moreover, the noncovalent binding of SUMO-interacting motif (SIM) at the SUMO surface was critically dependent on the paired aromatic interactions buried at the core. NMR structural studies revealed that perturbation of the Tyr51-Phe64 conformation disrupts several long-range tertiary contacts in SUMO, leading to a heterogeneous and dynamic protein with attenuated SUMOylation both in vitro and in cells. A subtle perturbation of the edge-to-face conformation by a Tyr to Phe substitution significantly decreased stability, SUMO/SIM affinity, and the rate of SUMOylation. Our results highlight that absolute co-conservation of specific aromatic pairs inside the SUMO protein core has a role in its stability and function.

Highlights

Aromatic amino acids buried at a protein’s core are often involved in mutual paired interactions
A conserved aromatic triad is present in the hydrophobic core of Small ubiquitin-like modifier (SUMO)
Reported high resolution (1.46 Å) crystal structures of human SUMO1 (PDB 4WJO) [34] shows that the tyrosine 51 (Tyr51) and phenylalanine 64 (Phe64) form an edge-to-face geometric orientation, where the edge of the Phe64 aromatic ring is perpendicular to the plane of the Tyr51 aromatic ring (Fig. 1, d and e)

Summary

Introduction

Aromatic amino acids buried at a protein’s core are often involved in mutual paired interactions. Ab initio energy calculations have highlighted that the conformational orientations and the effects of substitutions are important for stable aromatic interactions among aromatic rings, but studies in the context of a protein’s fold and function are elusive. We found that a specific edge-toface conformation between the Tyr51-Phe pair of interacting aromatics is vital to the fold and stability of SUMO. A subtle perturbation of the edge-to-face conformation by a Tyr to Phe substitution significantly decreased stability, SUMO/SIM affinity, and the rate of SUMOylation. Aromatic amino acids are hydrophobic and often present at the core of a protein, where the side chains are involved in paired interactions [6]. The atomistic details behind the stability of SUMO are essential to understand the SUMO pathway and SUMOylation-induced regulation of substrate stability

Methods

Results

Conclusion