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Intrinsic disorder drives N-terminal ubiquitination by Ube2w - PubMed

Intrinsic disorder drives N-terminal ubiquitination by Ube2w

Vinayak Vittal et al. Nat Chem Biol. 2015 Jan.

Abstract

Ubiquitination of the αN-terminus of protein substrates has been reported sporadically since the early 1980s. However, the identity of an enzyme responsible for this unique ubiquitin (Ub) modification has only recently been elucidated. We show the Ub-conjugating enzyme (E2) Ube2w uses a unique mechanism to facilitate the specific ubiquitination of the α-amino group of its substrates that involves recognition of backbone atoms of intrinsically disordered N termini. We present the NMR-based solution ensemble of full-length Ube2w that reveals a structural architecture unlike that of any other E2 in which its C terminus is partly disordered and flexible to accommodate variable substrate N termini. Flexibility of the substrate is critical for recognition by Ube2w, and either point mutations in or the removal of the flexible C terminus of Ube2w inhibits substrate binding and modification. Mechanistic insights reported here provide guiding principles for future efforts to define the N-terminal ubiquitome in cells.

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Figures

Figure 1
Figure 1

Ube2w has distinct E2 activity. (a) Ube2w transfers a single Ub to RPB8 in vitro while other BRCA1-interacting ubiquitin conjugating enzymes UbcH5c (“H5c”), UbcH7 (“H7”), and Ube2e1 (“e1”) do not (Supplementary Fig. 2a). (b) Left A nucleophile reactivity assay reveals Ube2w has intrinsic activity with αNH2 groups of a peptide with a free NH2 group at its N-terminus (NH2-A-G-G-S-Y-COO-; 50 mM) but not the εNH2 groups of lysine. Right, Identical reactions with UbcH5c~Ub conjugates confirm the previously reported lysine reactivity of UbcH5c and reveal it to be unreactive towards the peptide (Supplementary Fig. 2b). (c) Products generated on RPB8 depend on the Ub species in the reaction. Lanes 1 and 2: a single Ub is attached to RPB8 in a reaction with WT-Ub. Lane 3: Attachment of an additional Ub is detected in a reaction HA-Ub, which contains a 13-residue tag at the N-terminal end of Ub; Lane 4: Reaction carried out with lysine-less HA-Ub (HA-Ub(K0)) confirms that Ube2w builds linear Ub chains (i.e., attaches the C-terminus of one Ub to the N-terminus of another) on RPB8 with HA-Ub (Supplementary Fig. 2d). (d) Reactions shown in Panel (c) were blotted for Ub, revealing that Ube2w builds linear poly-Ub chains only when Ub harbors an N-terminal HA-tag (Supplementary Fig. 2e).

Figure 2
Figure 2

Ube2w transfers Ub to flexible/disordered N-termini. (a) Negative {1H – 15N} hetNOE values for residues derived from the HA-tag are indicative of highly flexible amino acids. Errors bars represent the standard error from the mean. (Resonances from this tag are not assigned and are not plotted sequentially. They are labeled simply as “HA”). (b) Consistent with the {1H – 15N} hetNOE data, the crystal structure of Ub (PDB: 1UBQ) is ordered at its αN-terminus and immediately forms a β -strand with residue Met1. (c) Ub to which two N-terminal amino acids have been added at the N-terminus (Met-Gly-Ub) is not incorporated into chains by Ube2w and displays similar activity to WT Ub. Four N-terminal residues (Met-Gly3-Ub) are sufficient to induce Ube2w activity towards Ub. Addition of six (Met-Gly5-Ub) or eight (Met-Gly7-Ub) residues increases Ube2w N-terminal ubiquitination activity (Note: bands below 37kDa are consistent with auto-ubiquitinated E2 and E3) (Supplementary Fig. 2f). (d) N-terminal backbone amide groups are necessary for Ube2w-dependent ubiquitination. Ube2w shows increased activity with the addition of disordered N-terminal amino acids, (Lanes 1-8). Proline at positions 2-4 (Lanes 9,10) inhibits Ube2w chain-building activity to levels similar to WT-Ub (Supplementary Fig. 2g).

Figure 3
Figure 3

The Ube2w C-terminus is flexible and occupies a non-canonical position. (a) Residues 7-132 of Ube2w have generally uniform and positive {1H – 15N} hetNOE values. Beginning at residue 137, values decrease and ultimately become negative at the extreme C-terminus, consistent with a region that undergoes motions at higher frequencies than the core of the protein. Errors bars represent the standard error from the mean. (b) For comparison, UbcH5c has positive hetNOE values throughout its entire protein sequence, even at the far C-terminus. (c) Left, Experimental CSP data based on comparing the (1H – 15N) – HSQC-TROSY spectra of Ube2w-KK and Ube2w-131Δ-KK reveals that removal of the C-terminus perturbs residues near the active site, in the 310-helix, and on the ‘backside’ β -sheet (purple). Right, If C-terminal helices were to reside in their canonical positions in Ube2w a surface consisting of loops 3 and 5 would be perturbed by removal of residues 132-151. Residues depicted to be perturbed are colored in purple, demonstrating that the C-terminal region of Ube2w is different from other E2s.

Figure 4
Figure 4

NMR ensemble of Ube2w reveals a novel E2 architecture. (a) Solution ensemble of Ube2w derived from NMR restraints (backbone chemical shifts, CSPs, residual dipolar couplings (RDCs), paramagnetic spin-label data, and small-angle X-ray scattering (SAXS)) calculated with CS-Rosetta. The twenty lowest energy members of the ensemble are shown and reveal a well-defined core with high structural similarity to canonical E2s. The C-terminal region is partially disordered and occupies multiple positions near the Ube2w active site C91 (orange). (b) Similar views of a representative canonical UBC domain structure (UbcH5c; PDB 2FUH). (c) Helix-4 (penultimate helix) is in distinct positions in Ube2w (3 representatives of the 20-member ensemble are shown for clarity). A flexible loop emanating from helix-3 leads away from the protein core. Helix-4 is clustered in three distinct positions in the ensemble (Cluster 1, light gray; Cluster 2, gray; Cluster 3, dark gray). (d) Side-view of the full Ube2w ensemble looking down the helix-3 axis reveals the three clusters. (e) In all twenty members of the Ube2w ensemble residues N136-W145 occupy positions beneath the active site, C91 (orange). Residues 119-135 are not shown for clarity. No clustering is evident for this region and the Cβ atom of every residue is on average 14.5-17.5 Å away from the active site.

Figure 5
Figure 5

The Ube2w C-terminus is required to interact with substrates. (a) {1H – 15N} – HSQC-TROSY spectrum of Ube2w-KK in the absence (black spectrum) and presence of 1 molar equivalent of RPB8 (red spectrum). Evidence for binding is seen as peak broadening (loss of intensity) and chemical shift perturbations of specific peaks in the Ube2w NMR spectrum. (b) A histogram showing chemical shift perturbations (CSPs) upon 1 molar equivalent of RP8 into Ube2w-KK. (c) Titration of 1 molar equivalent of tau into Ube2w-KK reveals very similar CSPs to addition of RPB8. (d) {1H – 15N} – HSQC-TROSY spectrum of Ube2w-131Δ-KK in the absence (black spectrum) and presence of 1 molar equivalent of RPB8 (red spectrum). Truncated Ube2w shows no interaction with RPB8 (e) Residues whose resonances have significant intensity losses and/or CSPs (> 1 standard deviation) are mapped in purple onto members of the Ube2w ensemble (one representative from each cluster). f) In an in vitro ubiquitination assay, Ube2w-131Δ does not transfer Ub to RPB8 after 1hr. Mutation of a single residue in the C-terminal region, W144E, also abrogates detectable activity. The loss of activity associated with the C-terminal region is equivalent to an active site-dead mutant, C91S (Supplementary Fig. 2k).

Figure 6
Figure 6

The Ube2w C-terminus facilitates α-amino group reactivity. (a) Selected {1H – 15N} resonances of residues near the Ube2w active site are compared in the spectra of full-length WT (black), W144E-Ube2w, (purple), and Ube2w-131Δ (red). Resonances move along similar trajectories as a result of the W144 mutation or C-terminal ablation, indicating similar chemical environments for the affected residues. (b) Mutation or ablation of the C-terminus affects the intrinsic aminolysis activity of Ube2w. In a 1hr reaction WT Ube2w~Ub shows robust transfer activity towards peptide (NH2-A-G-G-S-Y-COO-; 30 mM) as seen by increased amounts of free Ube2w and free Ub. Ube2w-W144E~Ub and the Ube2w-131Δ~Ub mutants show almost no Ub transfer activity to this minimal substrate over the same time period (Supplementary Fig. 2l).

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