Final Report Summary - BISSOT'D (Biochemical and structural characterisation of signal transduction by OTU family of Deubiquitinases)
Ubiquitination is a reversible protein modification that regulates diverse cellular functions including cell signalling, cell cycle, immune responses and gene expression. 8 differently linked polyubiquitin chains can be assembled using either 1 of the 7 lysine residues or Met1 in ubiquitin. Each linkage type adopts a distinct conformation and hence enzymes and ubiquitin binding domains have evolved ways to distinguish between different ubiquitin chain types and thereby achieve specificity. Protein ubiquitination is negatively regulated by specialised proteases called Deubiquitinases (DUBs) that reverse protein ubiquitination by hydrolysing the isopeptide linkage between ubiquitin moieties in ubiquitin chains or between ubiquitin and protein substrates. The ovarian tumour proteases (OTU) form a family of cysteine-based proteases and within the OTU family are the A20-like OTU DUBs consisting of A20, Cezanne1 and 2, VCIP135 and TRABID that have been shown to regulate ubiquitin signalling. Since there is no crystal structure of a cysteine protease in complex with a diubiquitin substrate, the exact catalytic mechanism employed is unclear. While A20 negatively regulates NF-kB signalling by removing K63-linked ubiquitin chains in cells, this OTU only cleaves K48-linked chains in vitro. Other members such as Cezanne1 and 2 show remarkable specificity towards K11 linkages, while TRABID hydrolyses K29 and K33 chains. Since Cezanne is highly selective for K11 linkages, we reasoned that obtaining a crystal structure of Cezanne in complex with K11-linked diubiquitin will provide insights into how substrate specificity is achieved. As a starting point, we solved the structure of the catalytic domain of Cezanne and this showed that it has a fold similar to that of the other A20-family OTUs. We then crystallised enzyme-product intermediate complexes by crystallising Cezanne in complex with ubiquitin. This structure reveals the distal ubiquitin binding site in Cezanne, which is distinct from that of other OTUs. We could confirm that the mode of distal ubiquitin binding is also similar in A20 by solving the structure of an A20~ubiquitin complex.
Comparing the apo and ubiquitin complex structures of Cezanne revealed that conformational changes take place to remodel the DUB from a closed catalytically incompetent conformation to an open active conformation where a proximal ubiquitin-binding site is created. Moreover, the different structures reveal that the catalytic centre of Cezanne is held in an inactive conformation in the resting state and can be remodelled to an active state upon ubiquitin binding.
By comparing the structures of A20 with that of TRABID and Cezane, we identified the existence of a loop in the catalytic domain of A20, which drives activity and specificity of A20. Based on our observations we hypothesise that this loop must be remodelled in order to activate the protease activity of A20. Further, we could show that the catalytic cysteine of A20 is regulated by reactive oxygen species (ROS) and this is a reversible regulation analogous to that observed in phosphatases. In summary, these findings reveal novel regulatory mechanisms modulating the activity of A20 and A20 family of OTU DUBs.
Comparing the apo and ubiquitin complex structures of Cezanne revealed that conformational changes take place to remodel the DUB from a closed catalytically incompetent conformation to an open active conformation where a proximal ubiquitin-binding site is created. Moreover, the different structures reveal that the catalytic centre of Cezanne is held in an inactive conformation in the resting state and can be remodelled to an active state upon ubiquitin binding.
By comparing the structures of A20 with that of TRABID and Cezane, we identified the existence of a loop in the catalytic domain of A20, which drives activity and specificity of A20. Based on our observations we hypothesise that this loop must be remodelled in order to activate the protease activity of A20. Further, we could show that the catalytic cysteine of A20 is regulated by reactive oxygen species (ROS) and this is a reversible regulation analogous to that observed in phosphatases. In summary, these findings reveal novel regulatory mechanisms modulating the activity of A20 and A20 family of OTU DUBs.