Dissecting Distinct Roles of NEDDylation E1 Ligase Heterodimer APPBP1 and UBA3 Reveals Potential Evolution Process for Activation of Ubiquitin-related Pathways.

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.

A linker strategy for trans-FRET assay to determine activation intermediate of NEDDylation cascade.

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.

Specific substrate recognition and thioester intermediate determinations in ubiquitin and SUMO conjugation cascades revealed by a high-sensitive FRET assay.

Ubiquitin and ubiquitin-like proteins (Ubls), such as SUMO, are covalently conjugated to their targets by related, but distinct enzymatic conjugation reactions that involve the dynamic E1-E2-E3 enzyme cascade. E1s activate Ubls by catalyzing Ubl C-terminal adenylation, with the help of ATP, to form a covalent thioester bond. Subsequently, Ubls are transferred to E2 to generate a thioester-linked product. In previous studies, we showed the dynamic processes and thioester intermediates of SUMO with its E1 and E2 conjugating enzymes. Studies of the enzyme specificity of the Ubl conjugation cascade are normally carried out by tedious biochemical processes, and the reaction intermediates are often difficult to capture because they are unstable and have short half-lives. Here, using our recently developed robust quantitative FRET-based technology, we describe systematic investigations of enzymatic specificity and thioester intermediate determination of ubiquitin with its E1-E2 ligases in conjugation with SUMO and its ligases. Our technology easily determined the strong specificity of enzyme-substrate interactions and thioester intermediates in ubiquitination and SUMOylation cascades. The traditional FRET pair ECFP/EYFP lacked adequate signals for these assays. However, in contrast, the highly sensitive FRET pair CyPet/YPet was easily harnessed to define the reaction specificities and intermediates. In addition, the thioester intermediates can be readily monitored by a newly defined FRET index parameter. These results provide an example of a systems biology approach to determine Ubl conjugation specificity and demonstrate that a robust FRET technology can be used to identify enzymes and substrates in other Ubl pathways.

Internal calibration Förster resonance energy transfer assay: a real-time approach for determining protease kinetics.

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research. This powerful tool can elucidate protein interactions in either a dynamic or steady state. We recently developed a series of FRET-based technologies to determine protein interaction dissociation constant and for use in high-throughput screening assays of SUMOylation. SUMO (small ubiquitin-like modifier) is conjugated to substrates through an enzymatic cascade. This important posttranslational protein modification is critical for multiple biological processes. Sentrin/SUMO-specific proteases (SENPs) act as endopeptidases to process the pre-SUMO or as isopeptidases to deconjugate SUMO from its substrate. Here, we describe a novel quantitative FRET-based protease assay for determining the kinetics of SENP1. Our strategy is based on the quantitative analysis and differentiation of fluorescent emission signals at the FRET acceptor emission wavelengths. Those fluorescent emission signals consist of three components: the FRET signal and the fluorescent emissions of donor (CyPet) and acceptor (YPet). Unlike our previous method in which donor and acceptor direct emissions were excluded by standard curves, the three fluorescent emissions were determined quantitatively during the SENP digestion process from onesample. New mathematical algorithms were developed to determine digested substrate concentrations directly from the FRET signal and donor/acceptor direct emissions. The kinetic parameters, kcat, KM, and catalytic efficiency (kcat/KM) of SENP1 catalytic domain for pre-SUMO1/2/3 were derived. Importantly, the general principles of this new quantitative methodology of FRET-based protease kinetic determinations can be applied to other proteases in a robust and systems biology approach.