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.
Subject(s)
Evolution, Molecular , NEDD8 Protein/chemistry , Ubiquitin-Activating Enzymes/chemistry , Fluorescence Resonance Energy Transfer , Humans , Molecular Dynamics Simulation , NEDD8 Protein/genetics , NEDD8 Protein/metabolism , Protein Binding , Protein Domains , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Static Electricity , Ubiquitin-Activating Enzymes/genetics , Ubiquitin-Activating Enzymes/metabolismABSTRACT
Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research and is a valuable tool for elucidating molecular interactions in vitro and in vivo. Quantitative FRET analysis is a powerful method for determining biochemical parameters and molecular distances at nanometer levels. Recently, we reported theoretical developments and experimental procedures for determining the dissociation constant, Kd and enzymatic kinetics parameters, Kcat and KM, of protein interactions with the engineered FRET pair, CyPet and YPet. The strong FRET signal from this pair made these developments possible. However, the direct link of fluorescent proteins with proteins of interests may interfere with the folding of some fusion proteins. Here, we report a new protein engineering strategy for improving FRET signals by adding a linker between the fluorescent protein and the targeted protein. This improvement allowed us to follow the covalent conjugation of NEDD8 to its E2 ligase in the presence of E1 and ATP, which was difficult to determine without linker. Three linkers, LAEAAAKEAA, TSGSPGLQEFGT, and LAAALAAA, which are alpha helix or random coil, all significantly improved the FRET signals. Our results show a general methodology for improving trans-FRET signals to effectively determine biochemical reaction intermediates.
