Abstract
Unanchored polyubiquitin chains are emerging as important regulators of cellular physiology with diverse roles paralleling those of substrate-conjugated polyubiquitin. However tools able to discriminate unanchored polyubiquitin chains of different isopeptide linkages have not been reported. We describe the design of a linker-optimized ubiquitin-binding domain hybrid (t-UBD) containing two UBDs, a ZnF-UBP domain in tandem with a linkage-selective UBA domain, which exploits avidity effects to afford selective recognition of unanchored Lys48-linked polyubiquitin chains. Utilizing native MS to quantitatively probe binding affinities we confirm cooperative binding of the UBDs within the synthetic protein, and desired binding specificity for Lys48-linked ubiquitin dimers. Furthermore, MS/MS analyses indicate that the t-UBD, when applied as an affinity enrichment reagent, can be used to favor the purification of endogenous unanchored Lys48-linked polyubiquitin chains from mammalian cell extracts. Our study indicates that strategies for the rational design and engineering of polyubiquitin chain-selective binding in nonbiological polymers are possible, paving the way for the generation of reagents to probe unanchored polyubiquitin chains of different linkages and more broadly the 'ubiquitome'. All MS data have been deposited in the ProteomeXchange with identifier PXD004059 (http://proteomecentral.proteomexchange.org/dataset/PXD004059).
Highlights
Ubiquitination, the covalent post-translational modification of target proteins with ubiquitin, is central to the regulation of a diverse array of biological processes [1]
3 Results 3.1 Designing and purifying the t-ubiquitin-binding domains (UBDs) We previously showed that the ZnF-UBP domain from the deubiquitinating enzyme USP5 recognises, with relatively high affinity, the free C-terminus of unanchored ubiquitin (Kd ≈ 2.3 μM vs monoubiquitin [22]), and can be used to affinity purify endogenous unanchored polyubiquitin chains from a range of biological sources [15, 16]
Subsequent quantitative electrospray ionisation mass spectrometry (ESI-MS) interaction studies confirmed that this UBD exhibits no intrinsic selectivity for unanchored ubiquitin dimers or tetramers of different isopeptide linkages [22]
Summary
Ubiquitination, the covalent post-translational modification of target proteins with ubiquitin, is central to the regulation of a diverse array of biological processes [1]. We previously used the isolated ZnF-UBP domain of USP5 to allow the affinity purification of unanchored polyubiquitin from a range of different species [15, 16], whilst alternative UBDs have been widely utilised to purify or detect ubiquitin-modified proteins in vitro [17], and to probe ubiquitin modifications in living cells [18], often with linkage selectivity. A future challenge is the development of bio-inspired synthetic reagents for the selective detection and purification of polyubiquitin chains of defined chain linkage/length, and which distinguish unanchored from conjugated polyubiquitin Towards this goal we demonstrate the rational design and validation using different MS approaches, of a tandem UBD (t-UBD) hybrid protein containing a ZnF-UBP domain joined to a Lys48polyubiquitin-selective UBA domain, which exploits avidity effects to allow the recognition and purification of unanchored Lys48-linked polyubiquitin chains. LCMS/MS was carried out using an RSLC nano HPLC system (Dionex, UK) and an LTQ-Orbitrap-Velos mass spectrometer (Thermo Scientific) (see supporting information for detail on sample loading and analysis)
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