Nonenzymatic rubylation and ubiquitination of proteins for structural and functional studies

Abstract

Uncovering the mechanisms that allow conjugates of ubiquitin (Ub) and/or Ub-like (UBL) proteins such as Rub1 to serve as distinct molecular signals requires the ability to make them with native connectivity and defined length and linkage composition. A novel, effective, and affordable strategy for controlled chemical assembly of fully natural UBL-Ub, Ub-UBL, and UBL-UBL conjugates from recombinant monomers is presented. Rubylation of Ub and Rub1 and ubiquitination of Rub1 was achieved without E2/E3 enzymes. New residue-specific information was obtained on the interdomain contacts in naturally-occurring K48-linked Rub1-Ub and Ub-Rub1, and K29-linked Rub1-Ub heterodimers, and their recognition by a K48-linkage-specific Ub receptor. The disassembly of these heterodimers by major deubiquitinating enzymes was examined and it was discovered that some deubiquitinases also possess derubylase activity. This unexpected result suggests possible crosstalk between Ub and Rub1/Nedd8 signaling pathways.

Keywords: deubiquitinases; nonenzymatic assembly; protein modifications; rubylation; ubiquitination.

Figure 1

Chemical assembly of Ub–⁴⁸Rub1, Rub1–⁴⁸Ub, and Rub1– ²⁹Ub dimers. (A) 2 mg of each reacting monomers, as indicated, were incubated in a condensation reaction for 36 hr. A 15% SDS-PAGE was performed with 2 μL of each reacting monomers, ligated product mix (non-purified), and the SEC-purified dimers/monomers. The running positions of monomers and dimers are indicated by the arrowhead and the arrow, respectively. (B-D) ESI-MS spectra of various intermediate and final steps during the assembly of Ub–⁴⁸Rub1 (B), Rub1–⁴⁸Ub (C), and Rub1–²⁹Ub (D) heterodimers. The molecular masses of the ¹⁵N-enriched Ub and Rub1 are 8,665 Da and 8,675 Da, respectively. The protection with the Boc group increases the mass by 100 Da, while the attachment of the SR group increases the mass by 125 Da.

Figure 2

Analysis of the interdomain interface in the heterodimers of Ub and Rub1. (A-B), ¹H-¹⁵N SOFAST-HMQC spectra (black) of Ub–⁴⁸Rub1 (A) and Rub1–²⁹Ub (B) overlaid with the spectra of Rub1 (red) and Ub (blue) monomers. Selected residues showing significant signal shifts are marked with numbers and indicated by arrows. Note that residue K48 in the proximal units was not ¹⁵N-labeled. (C, F), Chemical shift perturbations (CSP, black bars) and significant signal attenuations (>75%; grey bars) of backbone amides in Ub–⁴⁸Rub1 (C) and Rub1–²⁹Ub (F) plotted as a function of the residue number. Isopeptide- forming residues are connected by red arrows. (D-E, G-H), The perturbed residues (painted red) in Ub–⁴⁸Rub1 (D-E) and Rub1–²⁹Ub (G-H) are mapped on the 3D structures of Ub (blue) and Rub1 (grey), oriented such that the hydrophobic-patch surfaces face the reader except for (H) where the opposite side of Ub surface is shown.

Figure 3

K48-linked heterodimers of Rub1 and Ub form residue-specific interactions with the UBA2 domain of hHR23a. CSPs (black bars) and significant signal attenuations (>75%; grey bars) of backbone amides in Rub1–⁴⁸Ub (A) and Ub–⁴⁸Rub1 (D) at saturation with UBA2 are plotted as a function of the residue number. Residues with significant CSPs (>0.1 ppm) and signal attenuations are mapped (red) on the surface of the distal and proximal units of Rub1–⁴⁸Ub (B-C) and Ub–⁴⁸Rub1 (E-F). Ub and Rub1 are shown in blue and grey, respectively.

Figure 4

Cleavage of the synthesized heterodimers of Rub1 and Ub by deubiquitinases. The heterodimers were incubated for 16 hr in a cleavage reaction with indicated deubiquitinases in a 10:1 molar ratio. The reactions were stopped by adding SDS loading buffer and loaded onto a 15% SDS PAGE. The running positions of monomers and dimers are shown by the arrowhead and the arrow, respectively.

Scheme 1

Nonenzymatic rubylation of Ub. Similar schemes are used for ubiquitination or rubylation of Rub1. The basic steps involved in this method are as follows: (1) the C-terminus of either Rub1 or Ub (distal-to-be unit) is thioesterified (SR) by incubating the protein with its respective cognate E1 activating enzyme; (2) to direct the reaction to a specific lysine (e.g., K29) on the proximal-to-be unit (Ub or Rub1), the ε-amine of that lysine is protected with a Boc group (incorporated in the form of Lys(Boc) into the proximal-to-be unit as a genetically encoded unnatural amino acid (UAA); (3) all remaining free amines of both distal-to-be and proximal-to-be monomers are blocked with Alloc. Note that there are 12 such groups in distal-to-be Rub1 (8 lysines, 3 histidines, and the N-terminus) and 8 groups in proximal-to-be Ub (6 lysines, 1 histidine, and the N-terminus) which need to be protected with Alloc; (4) the Boc-group is removed to make the ε-amine of the lysine of interest the sole site for the ligation reaction; (5) ligation of the two monomers by Ag-mediated condensation reaction; (6) removal of all the Alloc groups from the product; followed by (7) renaturaton and separation of the dimer from the unreacted monomers, to yield a fully natural product, in this case Rub1–²⁹Ub. Boc=tert-butoxycarbonyl, Alloc=allyloxycarbonyl, MESNA=sodium 2-mercaptoethanesulfonate.