Mechanism of the Rpn13-induced activation of Uch37

Abstract

Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo-dimer of Uch37, each of the catalytic domains was blocking the other’s ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combination of mutagenesis, biochemical, NMR, and small-angle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiquitinating enzymes. Uch37ΔHb,Hc,KEKE, a truncation removal of the C-terminal extension region (residues 256–329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C (Rpn13 residues 270–407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 complex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-014-0046-z) contains supplementary material, which is available to authorized users.

Structural Basis for the Activation and Inhibition of the UCH37 Deubiquitylase

“…The RPN13 complex structure is consistent with the leading model that activation results from disruption of an inhibitory dimer (Jiao et al, 2014). UCH37 oligomerizes in a concentration-dependent manner and crystallizes in the absence of a binding partner as a dimer of dimers (Burgie et al, 2011).…”

“…UCH37 oligomerizes in a concentration-dependent manner and crystallizes in the absence of a binding partner as a dimer of dimers (Burgie et al, 2011). Although this point has been controversial, recent studies with SAXS and FRET have further reported that binding of RPN13 disrupts oligomerization to activate UCH37 (Jiao et al, 2014). This is attractive because binding of RPN13 and ubiquitin, as seen in our complex crystal structures, is incompatible with formation of the largest dimer interface seen in crystals of unliganded UCH37 (Figure 3A).…”

Meddling with Fate: The Proteasomal Deubiquitinating Enzymes

“…In the case of the proteasomal complex, Rpn13 DEU relieves autoinhibition of Uch37, which arises from two sources. First, the ULD domain of Uch37 is inhibitory to Uch37 activity [56,79,80], apparently because it can adopt conformations which sterically impede ubiquitin binding [60,61]. When bound to Rpn13 DEU , the ULD domain becomes fixed in an orientation that is permissive for substrate binding (Fig.…”

Protein Degradation Systems as Antimalarial Therapeutic Targets

“…Inhibitors of human Rpn13 inhibited proteasome function without inhibiting 20S catalytic activity or 19S deubiquitinating activity [60]. Rpn13 is a proteasome subunit that resides in the base of the 19S RP, binds K48-linked di-ubiquitin polypeptides via a pleckstrin-like receptor for ubiquitin (Pru) domain [61], and activates the proteasome-associated DUB UCH37 [62–64]. This strategy could potentially work in P. falciparum .…”