Crystal Structure of the Human Ubiquitin-like Protein NEDD8 and Interactions with Ubiquitin Pathway Enzymes

Frank G Whitby,Gang Xia,Cecile M Pickart,Christopher P Hill

doi:10.1074/jbc.273.52.34983

Frank G Whitby, Gang Xia + Show 2 more

Open Access

https://doi.org/10.1074/jbc.273.52.34983

Copy DOI

Abstract

The NEDD8/Rub1 class of ubiquitin-like proteins has been implicated in progression of the cell cycle from G1 into S phase. These molecules undergo a metabolism that parallels that of ubiquitin and involves specific interactions with many different proteins. We report here the crystal structure of recombinant human NEDD8 refined at 1.6-A resolution to an R factor of 21.9%. As expected from the high sequence similarity (57% identical), the NEDD8 structure closely resembles that reported previously for ubiquitin. We also show that recombinant human NEDD8 protein is activated, albeit inefficiently, by the ubiquitin-activating (E1) enzyme and that NEDD8 can be transferred from E1 to the ubiquitin conjugating enzyme E2-25K. E2-25K adds NEDD8 to a polyubiquitin chain with an efficiency similar to that of ubiquitin. A chimeric tetramer composed of three ubiquitins and one histidine-tagged NEDD8 binds to the 26 S proteasome with an affinity similar to that of tetraubiquitin. Seven residues that differ from the corresponding residues in ubiquitin, but are conserved between NEDD8 orthologs, are candidates for mediating interactions with NEDD8-specific partners. One such residue, Ala-72 (Arg in ubiquitin), is shown to perform a key role in selecting against reaction with the ubiquitin E1 enzyme, thereby acting to prevent the inappropriate diversion of NEDD8 into ubiquitin-specific pathways.

Highlights

Ubiquitin (Ub)1 is a small intracellular protein of 76 amino acid residues that is found both as a monomer and covalently conjugated to other protein molecules
We show that recombinant human NEDD8 protein is activated, albeit inefficiently, by the ubiquitin-activating (E1) enzyme and that NEDD8 can be transferred from E1 to the ubiquitin conjugating enzyme E2–25K
NEDD8 undergoes a metabolism which parallels that of Ub, including activation by a distinct E1-like enzyme (10, 17) and conjugation mediated by a dedicated E2 enzyme (10)

Summary

EXPERIMENTAL PROCEDURES

NEDD8 Plasmids—A human NEDD8-encoding gene comprising 76 amino acids was generated by PCR amplification using pcDNA/ hNEDD8 (14) as template. The purified inclusion bodies were suspended in buffer containing 50 mM Tris-HCl (24% base), 2 mM EDTA, and 8 M urea; the suspension was held at room temperature for 30 min, during which time it became clear. Thiol ester assays were carried out at pH 7.3 and 37 °C as described previously (28), using 4 ␮M labeled protein and ϳ3 ␮M E2. Degradation was monitored by the appearance of acid-soluble radioactivity and was linear in time and proteasome concentration The properties of this substrate, and the assay, will be described in detail elsewhere.. Prior to data collection the crystals were immersed for 2 (P21) or 30 (P1) min in a solution containing 100 mM citric acid, pH 4.8, 2.6 M ammonium sulfate, and 15% glycerol.

No of reflections observed

RESULTS AND DISCUSSION

Most favored