Structural and biophysical analysis of the proteasomal deubiquitinase, UCH37

Ubiquitin carboxyl-terminal hydrolase 37, or UCH37, is a deubiquitinating enzyme associated with the 26S proteasome, the primary protein degradation machinery in eukaryotic cells. UCH37 is responsible for the disassembly of polymeric ubiquitin chains, or polyubiquitin, which have been ligated onto proteins in order to target them for degradation. The 26S utilizes two associated deubiquitinating enzymes, UCH37 and USP14, and one intrinsic, Rpn11, to remove polyubiquitin chains from substrate proteins as they are unfolded and translocated into the proteolytic core of the proteasome, where proteins are cleaved into small peptides and then released for recycling by the cell. UCH37 associates with the proteasome via binding of its C-terminal KEKE motif to the C-terminus of Rpn13, a proteasomal ubiquitin receptor which ensnares polyubiquitinated prey for degradation. UCH37 is known to be catalytically activated upon binding to Rpn13, allowing cleavage of Lys48-linked polyubiquitin chains from their distal end, an exo-specific deubiquitination. However, free UCH37 cleaves polyubiquitin poorly and is believed to be autoinhibited by its C-terminal UCHL5-like domain, or ULD, which may also be responsible for its oligomerization in solution. This work examines the structural, biophysical, and catalytic characteristics of UCH37 in order to elucidate its mechanism of activation by Rpn13, assess its biophysical assembly with Rpn13 within the greater proteasomal context, and ascertain its mechanism of exo-specificity despite the proteasome\u27s processing of a variety of polyubiquitinated substrates. To this end, a 1.7 Å resolution x-ray crystal structure was solved of the catalytic domain of a UCH37 homolog from Trichinella spiralis in complex with ubiquitin vinyl methyl ester (UbVME), a suicide inhibitor substrate. Our structure, in combination with another solved of a longer construct of TsUCH37 in complex with UbVME, provided structural insights into the ability of UCH37 to process polyubiquitin, namely that its C-terminal UCHL5-like domain (ULD) is responsible for its exo-specific activity due to a network of interactions with ubiquitin\u27s Lys48. Through biophysical and kinetic characterization, we have affirmed the poor activity of UCH37 alone, but do not ascribe it to autoinhibition because it does not oligomerize as previously thought, rather we find that it sediments in a monomer-dimer equilibrium in analytical ultracentrifugation experiments. We have characterized its binding and activation by Rpn13, finding that UCH37 binds to Rpn13 with a 22 nM dissociation constant and that mutations to UCH37\u27s ULD render it unable to be activated by Rpn13. Interestingly, we have found that while Rpn13 activates UCH37 for ubiquitin-AMC cleavage, a monoubiquitin fluorogenic substrate, it appears to slow the enzyme\u27s processing of Lys48-linked polyubiquitin chains in our assays. Altogether, we have confirmed that UCH37 exists primarily as a monomer which binds tightly to its proteasomal subunit, Rpn13, and can exo-specifically cleave Lys48-linked polyubiquitin chains. However, UCH37 may not be activated as was previously thought, by Rpn13 alone, and likely requires full association with the 26S proteasome