Assembly and structure of Lys33-linked polyubiquitin reveals distinct conformations

Abstract

Ubiquitylation regulates a multitude of biological processes and this versatility stems from the ability of ubiquitin (Ub) to form topologically different polymers of eight different linkage types. Whereas some linkages have been studied in detail, other linkage types including Lys33-linked polyUb are poorly understood. In the present study, we identify an enzymatic system for the large-scale assembly of Lys33 chains by combining the HECT (homologous to the E6–AP C-terminus) E3 ligase AREL1 (apoptosis-resistant E3 Ub protein ligase 1) with linkage selective deubiquitinases (DUBs). Moreover, this first characterization of the chain selectivity of AREL1 indicates its preference for assembling Lys33- and Lys11-linked Ub chains. Intriguingly, the crystal structure of Lys33-linked diUb reveals that it adopts a compact conformation very similar to that observed for Lys11-linked diUb. In contrast, crystallographic analysis of Lys33-linked triUb reveals a more extended conformation. These two distinct conformational states of Lys33-linked polyUb may be selectively recognized by Ub-binding domains (UBD) and enzymes of the Ub system. Importantly, our work provides a method to assemble Lys33-linked polyUb that will allow further characterization of this atypical chain type.

Linkage-specific conformational ensembles of non-canonical polyubiquitin chains

“…CSPs were quantified as Δδ = [(Δδ H ) 2 + (Δδ N /5) 2 ] 1/2 , where Δδ H and Δδ N are the differences in 1 H and 15 N chemical shifts for the same residue between Ub 2 and monoUb. CSPs for K6-, K29-, and K33-Ub 2 s are in agreement with those recently published in 5,24–26 . Left panels show data for the distal Ub, right panels for the proximal Ub.…”

“…Interestingly, the Ub/Ub orientation in the major conformer of K29-Ub 2 ensembles is similar to that of K63-Ub 2 in complex with the tandem-UIM motif of Rap80 (Figure 13B). Moreover, as has been shown recently 24–26 , K29- and K33-Ub 2 can accommodate other binding partners, such as zinc-finger domain, by forming a sandwich-like complex that involves different residues on Ub beside the canonical hydrophobic patch. Other binding modes – yet to be determined – are also possible for any of the diubiquitins studied here.…”

“…20,21 ) and recent discoveries of several linkage-specific deubiquitinating enzymes 5 , crystal structures have been obtained for the unbound versions of some of these chains (Figure 1). 4,5,22–26 At the time of this writing, K27 remains the only lysine linkage for which there is no crystal structure. Importantly, the crystal structures (Figure 1) reveal that polyUb chains exist in multiple conformations.…”

A Rapid and Versatile Method for Generating Proteins with Defined Ubiquitin Chains

“…Several protocols have been published describing synthesis methods for lysine 29- and lysine 33-linked ubiquitin chains 35, 42 . These protocols can, in theory, be incorporated into the method described here by creating an appropriately blocked ubiquitin in the same fashion.…”

Peeling away the layers of ubiquitin signaling complexities with synthetic ubiquitin–protein conjugates

“…Instead, enzymatic or chemical methods are required to forge an isopeptide linkage. Enzymatic methods are powerful in their own right, but they offer limited control over which substrate lysine can be modified [10-15]. Chemical approaches, on the other hand, are relatively tunable and high yielding.…”