Abstract
Despite the similar enzyme cascade in the Ubiquitin and Ubiquitin-like peptide(Ubl) conjugation, the involvement of single or heterodimer E1 activating enzyme has been a mystery. Here, by using a quantitative Förster Resonance Energy Transfer (FRET) technology, aided with Analysis of Electrostatic Similarities Of Proteins (AESOP) computational framework, we elucidate in detail the functional properties of each subunit of the E1 heterodimer activating-enzyme for NEDD8, UBA3 and APPBP1. In contrast to SUMO activation, which requires both subunits of its E1 heterodimer AOS1-Uba2 for its activation, NEDD8 activation requires only one of two E1 subunits, UBA3. The other subunit, APPBP1, only contributes by accelerating the activation reaction rate. This discovery implies that APPBP1 functions mainly as a scaffold protein to enhance molecular interactions and facilitate catalytic reaction. These findings for the first time reveal critical new mechanisms and a potential evolutionary pathway for Ubl activations. Furthermore, this quantitative FRET approach can be used for other general biochemical pathway analysis in a dynamic mode.
Highlights
The conjugation of NEDD8, an Ubiquitin-like peptides, requires an enzymatic cascade, including E1, E2 and E3
The mutations in APPBP1 and UBA3 for the NEDD8 interface were selected by closely investigating the X-ray crystal structure (1R4N)[19], and mutations in APPBP1 for UBA3 interface and one mutation in UBA3 and NEDD8 were selected based on integrated Analysis of Electrostatic Similarities of Proteins (AESOP) analysis
Dissecting distinct roles of E1 heterodimer is critical for understanding Ubl activation in general
Summary
The conjugation of NEDD8, an Ubiquitin-like peptides, requires an enzymatic cascade, including E1, E2 and E3. Aided with Analysis of Electrostatic Similarities Of Proteins (AESOP) computational framework, we systematically dissected the reaction dynamics overtime, reaction intermediates, and both covalent and non-covalent molecular interactions of the three players, namely NEDD8, APPBP1 and UBA3, during NEDD8 activation in real time.
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