Molecular basis of Lys11-polyubiquitin specificity in the deubiquitinase Cezanne

The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types in polyubiquitin co-exist and are independently regulated in cells. This ‘ubiquitin code’ determines the fate of the modified protein1. Deubiquitinating enzymes of the Ovarian Tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin2, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system. We here reveal how the deubiquitinase Cezanne/OTUD7B specifically targets Lys11-linked polyubiquitin. Crystal structures of Cezanne alone and in complex with mono- and Lys11-linked diubiquitin, in combination with hydrogen-deuterium exchange mass spectrometry, enable reconstruction of the enzymatic cycle in exquisite detail. An intricate mechanism of ubiquitin-assisted conformational changes activate the enzyme, and while all chain types interact with the enzymatic S1 site, only Lys11-linked chains can bind productively across the active site and stimulate catalytic turnover. Our work highlights the fascinating plasticity of deubiquitinases, and indicates that new conformational states can occur when a true substrate, such as diubiquitin, is bound at the active site

Studying OTUD6B-OTUB1 protein-protein interaction by low-throughput GFP-trap assays and high-throughput AlphaScreen assays.

Protein-protein interactions (PPI) are involved in a myriad of cellular processes, and their deregulation can lead to many diseases. One such process is protein ubiquitination that requires an orchestrated action of three key enzymes to add ubiquitin moieties to substrate proteins. Importantly, this process is reversible through deubiquitinating enzymes. Both ubiquitination and deubiquitination require many PPIs that once classified can be utilized to identify small molecule inhibitors counteracting these reactions. Here, we study the protein-protein interaction between the two deubiquitinating enzymes OTUB1 and OTUD6B and report for the first time that both proteins directly interact with each other. We describe the GFP-Trap immunoprecipitation as a cell-based method to analyze the OTUD6B-OTUB1 interaction in the cellular context and the AlphaScreen (amplified luminescent proximity homogeneous assay) assay as a tool to detect direct interactions and to search for PPI inhibitors

Deubiquitinase-based analysis of ubiquitin chain architecture using Ubiquitin Chain Restriction (UbiCRest)

Protein ubiquitination is a versatile protein modification that regulates virtually all cellular processes. This versatility originates from polyubiquitin chains, which can be linked in eight distinct ways. The combinatorial complexity of eight linkage types in homotypic (one chain type per polymer) and heterotypic (multiple linkage types per polymer) chains poses significant problems for biochemical analysis. Here we describe UbiCRest, in which substrates (ubiquitinated proteins or polyubiquitin chains) are treated with a panel of linkage-specific deubiquitinating enzymes (DUBs) in parallel reactions, followed by gel-based analysis. UbiCRest can be used to show that a protein is ubiquitinated, to identify which linkage type(s) are present on polyubiquitinated proteins and to assess the architecture of heterotypic polyubiquitin chains. DUBs used in UbiCRest can be obtained commercially; however, we include details for generating a toolkit of purified DUBs and for profiling their linkage preferences in vitro. UbiCRest is a qualitative method that yields insights into ubiquitin chain linkage types and architecture within hours, and it can be performed on western blotting quantities of endogenously ubiquitinated proteins