Mechanism of the Rpn13-induced activation of Uch37

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 combi- nation 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 ubi- quitin-7-amino-4-methylcoumarin （Ub-AMC） of de-ubiq- uitinating enzymes. Uch37AHb＇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 com- plex. 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.