Profiling the Cross Reactivity of Ubiquitin with the Nedd8 Activating Enzyme by Phage Display

Bo Zhao,Jun Yin,Eric B Villhauer,Chan Hee J Choi,Heng Li,Karan Bhuripanyo,Hiroaki Kiyokawa,Keya Zhang,Hermann Schindelin,Ning Zheng

doi:10.1371/journal.pone.0070312

Abstract

The C-terminal peptides of ubiquitin (UB) and UB-like proteins (UBLs) play a key role in their recognition by the specific activating enzymes (E1s) to launch their transfer through the respective enzymatic cascades thus modifying cellular proteins. UB and Nedd8, a UBL regulating the activity of cullin-RING UB ligases, only differ by one residue at their C-termini; yet each has its specific E1 for the activation reaction. It has been reported recently that UAE can cross react with Nedd8 to enable its passage through the UB transfer cascade for protein neddylation. To elucidate differences in UB recognition by UAE and NAE, we carried out phage selection of a UB library with randomized C-terminal sequences based on the catalytic formation of UB∼NAE thioester conjugates. Our results confirmed the previous finding that residue 72 of UB plays a “gate-keeping” role in E1 selectivity. We also found that diverse sequences flanking residue 72 at the UB C-terminus can be accommodated by NAE for activation. Furthermore heptameric peptides derived from the C-terminal sequences of UB variants selected for NAE activation can function as mimics of Nedd8 to form thioester conjugates with NAE and the downstream E2 enzyme Ubc12 in the Nedd8 transfer cascade. Once the peptides are charged onto the cascade enzymes, the full-length Nedd8 protein is effectively blocked from passing through the cascade for the critical modification of cullin. We have thus identified a new class of inhibitors of protein neddylation based on the profiles of the UB C-terminal sequences recognized by NAE.

Highlights

Nedd8 is a ubiquitin-like protein (UBL) that covalently modifies the cullin subunits of the cullin-RING complexes to turn on their activities as E3 ubiquitin (UB) ligases (Figure 1a) [1,2,3,4,5,6]
We profiled the C-terminal sequences of UB that are catalytically active with Nedd8 activating enzyme (NAE) based on a phage display method we developed to engineer UB recognition by UAE (Figure 1b) [24]
The enriched phage clones were sequenced to identify the Cterminal sequences of UB variants that are reactive with NAE (Figure 1b)

Summary

Introduction

Nedd is a ubiquitin-like protein (UBL) that covalently modifies the cullin subunits of the cullin-RING complexes to turn on their activities as E3 ubiquitin (UB) ligases (Figure 1a) [1,2,3,4,5,6]. The E1 enzyme specific for Nedd, known as Nedd activating enzyme (NAE), catalyzes the condensation of ATP with the Cterminal carboxylate of Nedd to form a Nedd8-AMP conjugate [5,7,8]. A thioester exchange reaction leads to the transfer of Nedd from NAE to the E2 enzyme that carries Nedd to cullin for its modification [9]. UB has its own set of one or two E1s and several dozen E2s that activate and transfer UB following the same mechanism; the E1 enzymes specific for UB (UB activation enzyme or UAE) catalyze the formation of UB,E1 conjugates followed by UB transfer to E2s to form UB,E2 conjugates. The UB,E2 conjugates are bound to the E3 enzymes such as the cullin-RING complexes to deliver UB to the substrate proteins recruited by the E3s [10,11]

Methods

Results

Conclusion