Biochemical and structural characterization of a novel ubiquitin-conjugating enzyme E2 from Agrocybe aegeria reveals Ube2w family-specific properties.

Chao Qi,Hui Sun,Yanjie Hou,De-Feng Li,Da-Cheng Wang,Wei Liu,Lei Feng

doi:10.1038/srep16056

Abstract

Ubiquitination is a post-translational modification that is involved in myriad cellar regulation and disease pathways. The ubiquitin-conjugating enzyme (E2) is an important player in the ubiquitin transfer pathway. Although many E2 structures are available, not all E2 families have known structures, and three-dimensional structures from fungal organisms other than yeast are lacking. We report here the crystal structure of UbcA1, which is a novel ubiquitin-conjugating enzyme identified from the edible and medicinal mushroom Agrocybe aegerita and displays potential antitumor properties. The protein belongs to the Ube2w family and shows similar biochemical characteristics to human Ube2w, including monomer-dimer equilibrium in solution, α-NH2 ubiquitin-transfer activity and a mechanism to recognize backbone atoms of intrinsically disordered N-termini in substrates. Its structure displays a unique C-terminal conformation with an orientation of helix α3 that is completely different from the reported E2 structures but similar to a recently reported NMR ensemble of Ube2w. A mutagenesis study on this novel enzyme revealed that an intact C-terminus is significant for protein dimerization and enzymatic activity. As the first crystallized full-length protein of this family, UbcA1 may supersede the truncated X-ray structure of Ube2w (PDB entry 2A7L) as the representative structure of the Ube2w family.

Highlights

160 structures classified in the CATH ubiquitin-conjugate enzyme superfamily (3.10.110.10) have been deposited in the Protein Data Bank (PDB), most of which are of an individual Ubc domain
We describe here the biochemical and structural characterization of this novel Ub-conjugating enzyme, which is the first fungal E2 enzyme other than the homologues from yeast crystallized to date
UbcA1 belongs to the Ube2w family

Summary

Introduction

All E2 enzymes are characterized by the presence of a highly conserved ubiquitin-conjugating (Ubc) domain comprising ~150 amino acids and adopting a similar overall fold[14]. Critical mechanical elements include the active site cysteine nestled in a shallow groove close to the 310-helix, an upstream ‘HPN’ tripeptide and a downstream highly conserved tryptophan that often contacts a conserved proline residue located on L411,13. In addition to these common features, the Ubc fold represents a rare example in enzyme evolution with high structural diversity[11], and even the Ubc domain alone can be classified into 17 subfamilies[12]. We describe here the biochemical and structural characterization of this novel Ub-conjugating enzyme, which is the first fungal E2 enzyme other than the homologues from yeast crystallized to date

Methods

Results

Conclusion