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

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.

Figure 1

Subunit of E1, Uba3, alone is sufficient for NEDD8 activation. (A) FRET-based protein assay for determining the E1 requirement of APPBP1 and UBA3 for NEDD8 activation and conjugation to Ubc12(data are represented as mean +/− SD). The Time in x-axis represents the time of components mixing time. (B) Neddylation assay by Western-blot analysis. The conjugation of NEED8 to Ubc12 was performed with or without APPBP1 at different time points and blotted with anti-NEEd8 antibody.

Figure 2

APPBP1-ATP binding activity contributes, but is not required for NEDD8 activation. (A) Close-up view of the ATP-binding pocket in E1 heterodimer of NEDD8 (PDB ID: 1R4N), UBA3 shown in red, APPBP1 in blue, NEDD8 in green, ATP in magenta, residue from UBA3 participating in ATP binding pocket in orange and from APPBP1 in cyan (parts of APPBP1 and UBA3 are hidden for clarity). (B) APPBP1’s ATP-binding residue (Arg15) is not critical for NEDD8 conjugation to Ubc12 (data are represented as mean +/− SD).

Figure 3

Non-covalent interactions between APPBP1 and NEDD8 are not critical for NEDD8 activation and conjugation. (A–C) FRET based protein assay for determining importance of PPBP1 and NEDD8 non-covalent interface for NEDD8 activation and conjugation to Ubc12. (D) Effects of mutations in APPBP1 on APPBP1-NEDD8 non-covalent interactions (data are represented as mean +/− SD).

Figure 4

Non-covalent interactions between UBA3 and NEDD8 are critical for NEDD8 activation and conjugation. (A) Effects of mutations in UBA3 on UBA3-NEDD8 non-covalent interactions (data are represented as mean +/− SD). (B) FRET-based assay of NEDD8 and Uba3V344A mutant in NEED8 activation and conjugation. (C) FRET-based assay of NEDD8 and Uba3V344AY352A double mutant in NEDD8 activation and conjugation. (D) FRET-based assay of NEDD8 and Uba3V344AY352AE366A triple mutant in NEDD8 activation and conjugation.

Figure 5

Non-covalent interactions between APPBP1 and UBA3 are important, but required, for NEDD8 activation and conjugation. (A) Alanine scan electrostatic clustering and free energies of association. Clustering dendrogram of the alanine scan mutants of APPBP1 using the average weighted difference ESD. Free energy of mutants ordered according to average weighted difference clustering. Non-covalent interactions between APPBP1 and UBA3 are partially critical for NEDD8 activation and conjugation. (B) FRET-based assay for interactions of APPBP1 mutants with UBA3 (data are represented as mean +/−SD). (C–E) FRET-based NEED8 activation assay for APPBP1 mutants disrupting interaction with Uba3.

Figure 6

Comparison of activating enzymes for Ubiquitin and Ubl proteins with those for the MoaD and ThiF. The four Ubls whose E1s are most closely related are ubiquitin, SUMO, NEDD8 and ISG15 and their family members. The Ubl E1s are 110–120 kDa and are either single polypeptides or heterodimers of two polypeptides, corresponding to the sequence and structure of MoeB. The E1s of ubiquitin and ISG15 are single polypeptides. In case of heterodimeric E1s (SUMO and NEDD8 cascade), one subunit corresponds to N terminal half of the single-chain E1s and other subunit of the heterodimer corresponds to C-terminal of half. The N- and C-terminal halves of the E1s for ubiquitin, SUMO, NEDD8 and ISG15 are partially homologous to each other, and the region of sequence homology between the two halves is also the region of sequence homology to MoeB and ThiF.