Mechanisms of protein recognition have been extensively studied for single-domain proteins1, but are less well characterized for dynamic multi-domain systems. Ubiquitin (Ub) chains represent a biologically important multi-domain system that requires recognition by structurally diverse Ub-interacting proteins (UbIPs)2,3. Ub chain conformations in isolation are often different from conformations observed in UbIP complexes, suggesting either great dynamic flexibility or extensive chain remodeling upon binding.Using single-molecule FRET, we show here that Lys63-, Lys48- and Met1-linked diUb exist in several distinct conformational states in solution. Lys63- and Met1-linked diUb adopt extended ‘open’ and more compact ‘closed’ conformations, and Ub binding domains (UBDs) and deubiquitinases (DUBs) select pre-existing conformations. In contrast, Lys48-linked diUb adopts predominantly compact conformations. DUBs directly recognize existing conformations, but may also remodel Ub chains to hydrolyze the isopeptide bond. Disruption of the Lys48-diUb interface changes conformational dynamics and affects DUB activity. Hence, conformational equilibria in Ub chains provide an additional layer of regulation in the Ub system, and distinct conformations observed in differently linked polyUb may contribute to the specificity of UbIPs.

Crimean Congo hemorrhagic fever virus (CCHFV) is a deadly human pathogen that evades innate immune responses by efficiently interfering with antiviral signaling pathways mediated by NF-κB, IRF3, and IFNα/β. These pathways rely on protein ubiquitination for their activation, and one outcome is the modification of proteins with the ubiquitin (Ub)-like modifier interferon-stimulated gene (ISG)15. CCHFV and related viruses encode a deubiquitinase (DUB) of the ovarian tumor (OTU) family, which unlike eukaryotic OTU DUBs also targets ISG15 modifications. Here we characterized the viral OTU domain of CCHFV (vOTU) biochemically and structurally, revealing that it hydrolyzes four out of six tested Ub linkages, but lacks activity against linear and K29-linked Ub chains. vOTU cleaved Ub and ISG15 with similar kinetics, and we were able to understand vOTU cross-reactivity at the molecular level from crystal structures of vOTU in complex with Ub and ISG15. An N-terminal extension in vOTU not present in eukaryotic OTU binds to the hydrophobic Ile44 patch of Ub, which results in a dramatically different Ub orientation compared to a eukaryotic OTU-Ub complex. The C-terminal Ub-like fold of ISG15 (ISG15-C) adopts an equivalent binding orientation. Interestingly, ISG15-C contains an additional second hydrophobic surface that is specifically contacted by vOTU. These subtle differences in Ub/ISG15 binding allowed the design of vOTU variants specific for either Ub or ISG15, which will be useful tools to understand the relative contribution of ubiquitination vs. ISGylation in viral infection. Furthermore, the crystal structures will allow structure-based design of antiviral agents targeting this enzyme.cell signaling | structural biology | biochemistry | emerging disease

Ubiquitin specific proteases (USPs) are the largest family of deubiquitinating enzymes with approximately 56 members in humans. USPs regulate a wide variety of cellular processes by their ability to remove (poly)ubiquitin from target proteins. Their enzymatic activity is encoded in a common catalytic core of approximately 350 amino acids, however many USPs show significantly larger catalytic domains. Here we have analysed human and yeast USP domains, combining bioinformatics with structural information. We reveal that all USP domains can be divided into six conserved boxes, and we map the conserved boxes onto the USP domain core structure. The boxes are interspersed by insertions, some of which as large as the catalytic core. The two most common insertion points place inserts near the distal ubiquitin binding site, and in many cases ubiquitin binding domains or ubiquitin-like folds are found in these insertions, potentially directly affecting catalytic function. Other inserted sequences are unstructured, and removal of these might aid future structural and functional analysis. Yeast USP domains have a different pattern of inserted sequences, suggesting that the insertions are hotspots for evolutionary diversity to expand USP functionality.

SummaryThe endosomal sorting complexes required for transport (ESCRTs) facilitate endosomal sorting of ubiquitinated cargo, MVB biogenesis, late stages of cytokinesis, and retroviral budding. Here we show that ubiquitin associated protein 1 (UBAP1), a subunit of human ESCRT-I, coassembles in a stable 1:1:1:1 complex with Vps23/TSG101, VPS28, and VPS37. The X-ray crystal structure of the C-terminal region of UBAP1 reveals a domain that we describe as a solenoid of overlapping UBAs (SOUBA). NMR analysis shows that each of the three rigidly arranged overlapping UBAs making up the SOUBA interact with ubiquitin. We demonstrate that UBAP1-containing ESCRT-I is essential for degradation of antiviral cell-surface proteins, such as tetherin (BST-2/CD317), by viral countermeasures, namely, the HIV-1 accessory protein Vpu and the Kaposi sarcoma-associated herpesvirus (KSHV) ubiquitin ligase K5.