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. 2013 Jun 15;21(12):3421-9.
doi: 10.1016/j.bmc.2013.02.052. Epub 2013 Mar 15.

Nonenzymatic assembly of branched polyubiquitin chains for structural and biochemical studies

Emma K Dixon  1 , Carlos A Castañeda, Tanuja R Kashyap, Yan Wang, David Fushman
Affiliations

Nonenzymatic assembly of branched polyubiquitin chains for structural and biochemical studies

Emma K Dixon et al. Bioorg Med Chem. .

Abstract

Polymeric chains of a small protein ubiquitin are involved in regulation of nearly all vital processes in eukaryotic cells. Elucidating the signaling properties of polyubiquitin requires the ability to make these chains in vitro. In recent years, chemical and chemical-biology tools have been developed that produce fully natural isopeptide-linked polyUb chains with no need for linkage-specific ubiquitin-conjugating enzymes. These methods produced unbranched chains (in which no more than one lysine per ubiquitin is conjugated to another ubiquitin). Here we report a nonenzymatic method for the assembly of fully natural isopeptide-linked branched polyubiquitin chains. This method is based on the use of mutually orthogonal removable protecting groups (e.g., Boc- and Alloc-) on lysines combined with an Ag-catalyzed condensation reaction between a C-terminal thioester on one ubiquitin and a specific ε-amine on another ubiquitin, and involves genetic incorporation of more than one Lys(Boc) at the desired linkage positions in the ubiquitin sequence. We demonstrate our method by making a fully natural branched tri-ubiquitin containing isopeptide linkages via Lys11 and Lys33, and a (15)N-enriched proximal ubiquitin, which enabled monomer-specific structural and dynamical studies by NMR. Furthermore, we assayed disassembly of branched and unbranched tri-ubiquitins as well as control di-ubiquitins by the yeast proteasome-associated deubiquitinase Ubp6. Our results show that Ubp6 can recognize and disassemble a branched polyubiquitin, wherein cleavage preferences for individual linkages are retained. Our spectroscopic and functional data suggest that, at least for the chains studied here, the isopeptide linkages are effectively independent of each other. Together with our method for nonenzymatic assembly of unbranched polyubiquitin, these developments now provide tools for making fully natural polyubiquitin chains of essentially any type of linkage and length.

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Figures

Figure 1
Figure 1
Assembly of [Ub]2–11,33Ub. (A) SDS-PAGE of the ligation reaction. Ub-COSRA is a fully Alloc-protected Ub containing a C-terminal thioester. 15N K11,K33 UbA is Ub that is Allocprotected except for K11 and K33, where the genetically-incorporated Boc protecting groups have been removed via TFA treatment. After 16 hours, ligation was complete and shows a tri-Ub band. (B) ESI-MS analysis of the 15N Ub K11Boc,K33Boc protein prior to Alloc protection or TFA treatment. The observed molecular weight is 8,860 Da, consistent with a 15N-enriched Ub containing two 14N-labeled Lys(Boc) amino acids. While the MS data also indicate the presence of a Ub species containing a single Boc group, this protein would terminate at the dimer stage during a ligation reaction. (C,D) Size-exclusion chromatography of fully Alloc-deprotected and renatured ligation product, indicating separation of tri-Ub from incompletely reacted Ub2 and Ub. (E) [Ub]2–11,33Ub is >95% by SDS-PAGE. (F) ESI-MS analysis of purified [Ub]2–11,33Ub, indicating a molecular weight of 25,755 Da, consistent with a tri-Ub species containing one 15N Ub, two 14N Ub units, and the loss of two H2O molecules upon isopeptide bonds formation.
Figure 2
Figure 2
NMR characterization of [Ub]2–11,33Ub and Ub K11Boc,K33Boc. (A) Overlay of 1H-15N TROSY spectra of the proximal Ub in [Ub]2–11,33Ub (red) and of monomeric wild type Ub (blue). (B) Overlay of 1H-15N TROSY spectra of Ub K11Boc,K33Boc monomer (red) and of wild type Ub (blue). Note the absence of (red) NMR signals for residues 11 and 33 in A and B due to the incorporation of unlabeled Lys(Boc) at these positions.
Figure 3
Figure 3
Spectral differences, quantified as amide chemical shift perturbations (CSPs, Δδ), of the proximal Ub in [Ub]2–11,33Ub versus (A) monoUb, (C) the proximal Ub in K11-linked Ub2, and (E) the proximal Ub in K33-linked Ub2. (B) CSPs of Ub K11Boc,K33Boc monomer versus Ub. (D,F) CSPs of the proximal Ub in K33-linked Ub2 (D) and proximal Ub in K11-linked Ub2 (F) versus Ub. Absent NMR signals due to the incorporation of unlabeled Lys(Boc) are marked with asterisks.
Figure 4
Figure 4
15N T1 relaxation time measured for backbone amides in the proximal Ub of [Ub]2–11,33Ub (black) and in the proximal Ub in K11-linked Ub2 (green). The dashed horizontal lines represent the average levels of 15N T1 for residues in secondary structure elements.
Figure 5
Figure 5
Disassembly of polyubiquitin chains by a proteasome-associated deubiquitinase Ubp6. A cartoon representation of each polyUb species is depicted on the left side of the corresponding gel.
Scheme 1
Scheme 1
Non-enzymatic assembly of K11,K33 branched tri-Ub ([Ub]2–11,33Ub). Gray shading indicates Ub unit that is 15N-enriched. Asterisks (*) represent the Alloc protecting group.
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