Familial and Somatic BAP1 Mutations Inactivate ASXL1/2-Mediated Allosteric Regulation of BAP1 Deubiquitinase by Targeting Multiple Independent Domains

Abstract

Deleterious mutations of the ubiquitin carboxy-terminal hydrolase BAP1 found in cancers are predicted to encode inactive truncated proteins, suggesting that loss of enzyme function is a primary tumorigenic mechanism. However, many tumors exhibit missense mutations or in-frame deletions or insertions, often outside the functionally critical UCH domain in this tumor suppressor protein. Thus, precisely how these mutations inactivate BAP1 is unknown. Here, we show how these mutations affect BAP1 interactions with the Polycomb group-like protein, ASXL2, using combinations of computational modeling technology, molecular biology, and reconstitution biochemistry. We found that the BAP1-ASXL2 interaction is direct and high affinity, occurring through the ASXH domain of ASXL2, an obligate partner for BAP1 enzymatic activity. The ASXH domain was the minimal domain for binding the BAP1 ULD domain, and mutations on the surfaces of predicted helices of ASXH abolished BAP1 association and stimulation of BAP1 enzymatic activity. The BAP1-UCH, BAP1-ULD, and ASXH domains formed a cooperative stable ternary complex required for deubiquitination. We defined four classes of alterations in BAP1 outside the UCH domain, each failing to productively recruit ASXH to the wild-type BAP1 catalytic site via the ULD, resulting in loss of BAP1 ubiquitin hydrolase activity. Our results indicate that many BAP1 mutations act allosterically to inhibit ASXH binding, thereby leading to loss of enzyme activity. Small-molecule approaches to reactivate latent wild-type UCH activity of these mutants might be therapeutically viable. Combined computational and biochemical approaches demonstrate that the BAP1-ASXL2 interaction is direct and high affinity and that many mutations act allosterically to inhibit BAP1-ASXL2 binding..

Kinetics characterization of ASXL1/2-mediated allosteric regulation of BAP1 deubiquitinase

“…1B-C). Using the structure of Drosophila Calypso UCH/ULD interaction with Asx (PDB 6HGC) converted into human BAP1 UCH/ULD and ASXL2 merged with our previous models of interaction with H2A and Ubiquitin (16), we can pinpoint the human contact maps of the ULD with ASXL2 with high confidence (Fig. 1D, E).…”

“…From our previous studies (16), we learned that the BAP1-ULD domain interacts directly with the ASXL2-AB box. However, the binding kinetics and stoichiometry of interaction of the ULD domain and the AB box remained unknown.…”

“…Cancer-related mutations/deletions of BAP1 often result in loss-of-function by causing premature protein termination, protein instability and/or loss of UCH catalytic activity. Other mutations of BAP1 lead to loss-of-function by targeting the ULD domain, thereby disrupting binding to ASXL2 (16) an obligate partner for BAP1 enzymatic activity.…”

“…Calypso interacts with PcG protein Asx , and this Polycomb repressive deubiquitinase (PR-DUB) complex binds to PcG target genes. The human homologs of Asx are ASXL1-3 (16). The N-terminus of ASXL contains the highly conserved Asx homology domain (ASXH), which is required for Calypso/BAP1 protein binding.…”

Twist, Snail, and Sox9 form an allosterically regulated complex, the EMTosome, on a bipartite E-box site

“…Immunoprecipitations and Western Blots were done as previously described except as noted below (19). Addition of oligonucleotides to NE increased affinity to flag antibody resin.…”