Conservation in the Mechanism of Nedd8 Activation by the Human AppBp1-Uba3 Heterodimer

Richard N Bohnsack,Arthur L Haas

doi:10.1074/jbc.m303177200

Abstract

Human Nedd8-activating enzyme AppBp1-Uba3 was purified to apparent homogeneity from erythrocytes. In the presence of [2,8-3H]ATP and 125I-Nedd8, heterodimer rapidly forms a stable stoichiometric ternary complex composed of tightly bound Nedd8 [3H]adenylate and Uba3-125I-Nedd8 thiol ester. Isotope exchange kinetics show that the heterodimer follows a pseudo-ordered mechanism with ATP the leading and Nedd8 the trailing substrate. Human AppBp1-Uba3 follows hyperbolic kinetics for HsUbc12 transthiolation with 125I-Nedd8 (kcat = 3.5 +/- 0.2 s-1), yielding Km values for ATP (103 +/- 12 microm), 125I-Nedd8 (0.95 +/- 0.18 microm), and HsUbc12 (43 +/- 13 nm) similar to those for ubiquitin activation by Uba1. Wild type 125I-ubiquitin fails to support AppBp1-Uba3 catalyzed activation or HsUbc12 transthiolation. However, modest inhibition of 125I-Nedd8 ternary complex formation by unlabeled ubiquitin suggests a Kd > 300 microm for ubiquitin. Alanine 72 of Nedd8 is a critical specificity determinant for AppBp1-Uba3 binding because 125I-UbR72L undergoes heterodimer-catalyzed hyperbolic HsUbc12 transthiolation and yields Km = 20 +/- 9 microm and kcat = 0.9 +/- 0.3 s-1. These observations demonstrate remarkable conservation in the mechanism of AppBp1-Uba3 that mirrors its sequence conservation with the Uba1 ubiquitin-activating enzyme.

Highlights

Class I ubiquitin-like proteins exert their biological effects through covalent conjugation to their respective target proteins via distinct ligation pathways that function in parallel to those of ubiquitination
In the activation of ubiquitin, Uba1 forms a ternary complex composed of 1 eq each of a tightly bound ubiquitin adenylate and a covalent Uba1-ubiquitin thiol ester to a conserved active site, Cys632 [28, 29], HsUba1a numbering
The Uba1 ubiquitin-activating enzyme catalyzes the first step in the conjugation of ubiquitin to protein targets and serves as the archetype for similar steps in the activation of other ubiquitin paralogs that include Sumo, Nedd8, Hub1, ISG15, FAT10, and Apg12 [10]

Summary

Introduction

Class I ubiquitin-like proteins exert their biological effects through covalent conjugation to their respective target proteins via distinct ligation pathways that function in parallel to those of ubiquitination. The recent 2.6 Å structure of human AppBp1-Uba confirms that AppBp1 is required in part to contribute a short conserved active site segment first identified in the mechanistically related MoeB subunit of molybdopterin synthase [21, 27]. The apparently substoichiometric formation of the predicted Nedd adenylate intermediate catalyzed by the reconstituted plant ortholog of AppBp1-Uba suggests that the catalytic cycle for Nedd activation may exhibit some differuitin carrier protein or ubiquitin-conjugating enzyme (Ubc); GST, glutathione S-transferase; DTT, dithiothreitol; Ub, ubiquitin. Mechanism of Nedd Activation ences from that of ubiquitin [30] The latter observation is significant because the presence of enzyme-bound ubiquitin adenylate is required for ubiquitin transthiolation from the E1 ternary complex to E2 carrier proteins, even though this intermediate is not the immediate donor of activated polypeptide [31]. The enzymes involved in the activation of ubiquitin-like proteins have not been mechanistically characterized in sufficient detail to resolve these and related questions

Methods

Results

Conclusion