Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15

Abstract

Highlights d Tight and selective UbVs target USP15 catalytic and adaptor domains d UbV inhibitors lock the USP15 active site in an inactive conformation d A strand-swapped UbV dimer binds two DUSP domains simultaneously d Linear UbV dimers are potent and specific USP15 inhibitors in cells SUMMARYThe multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor b pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation.

Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site

“…Interestingly, the domain-swapping of UbV W is reminiscent of that recently reported for other UbVs, one that binds the dimeric RING-RING assembly from the E3 XIAP (UbV.XR) and another that binds the DUSP domain of the DUB USP15 (UbV.15.D) (42,50). Conformational differences between 2 independent crystal structures of UbV.XR suggested flexibility in the β-sheet formed by the domain swap, with different relative orientations varying by 30°for the 2 halves of the dimer.…”

USP15 deubiquitinase safeguards hematopoiesis and genome integrity in hematopoietic stem cells and leukemia cells

“…Our data strongly argue in favor of developing specific USP15 inhibitors, which are only starting to emerge. Interestingly, engineered ubiquitin variants targeting USP15 have recently been reported (Teyra et al, 2019), which may open up to the development of more specific small molecules as well as targeted degraders. More broadly, our study calls for a more systematic effort in understanding how DUBs regulate normal and malignant HSC biology as a critical route towards the selection of effective drug targets and targeted treatment combinations.…”

Ubiquitin in disguise unveils a cryptic binding site in 1.2-MDa anaphase-promoting complex/cyclosome

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