Proapoptotic BH3-interacting death domain agonist (BID) regulates apoptosis and the DNA damage response. Following replicative stress, BID associates with proteins of the DNA damage sensor complex, including replication protein A (RPA), ataxia telangiectasia and Rad3 related (ATR), and ATR-interacting protein (ATRIP), and facilitates an efficient DNA damage response. We have found that BID stimulates the association of RPA with components of the DNA damage sensor complex through interaction with the basic cleft of the N-terminal domain of the RPA70 subunit. Disruption of the BID-RPA interaction impairs the association of ATR-ATRIP with chromatin as well as ATR function, as measured by CHK1 activation and recovery of DNA replication following hydroxyurea (HU). We further demonstrate that the association of BID with RPA stimulates the association of ATR-ATRIP to the DNA damage sensor complex. We propose a model in which BID associates with RPA and stimulates the recruitment and/or stabilization of ATR-ATRIP to the DNA damage sensor complex.

REV1 is a DNA damage tolerance protein and encodes two ubiquitin-binding motifs (UBM1 and UBM2) that are essential for REV1 functions in cell survival under DNA-damaging stress. Here we report the first solution and X-ray crystal structures of REV1 UBM2 and its complex with ubiquitin, respectively. Furthermore, we have identified the first small-molecule compound, MLAF50, that directly binds to REV1 UBM2. In the heteronuclear single quantum coherence NMR experiments, peaks of UBM2 but not of UBM1 are significantly shifted by the addition of ubiquitin, which agrees to the observation that REV1 UBM2 but not UBM1 is required for DNA damage tolerance. REV1 UBM2 interacts with hydrophobic residues of ubiquitin such as L8 and L73. NMR data suggest that MLAF50 binds to the same residues of REV1 UBM2 that interact with ubiquitin, indicating that MLAF50 can compete with the REV1 UBM2-ubiquitin interaction orthosterically. Indeed, MLAF50 inhibited the interaction of REV1 UBM2 with ubiquitin and prevented chromatin localization of REV1 induced by cisplatin in U2OS cells. Our results structurally validate REV1 UBM2 as a target of a small-molecule inhibitor and demonstrate a new avenue to targeting ubiquitination-mediated protein interactions with a chemical tool.

E3 ligase recruitment of proteins containing terminal destabilizing motifs (degrons) is emerging as a major form of regulation. How those E3s discriminate bona fide substrates from other proteins with terminal degron-like sequences remains unclear. Here, we report that human KLHDC2, a CRL2 substrate receptor targeting C-terminal Gly-Gly degrons, is regulated through interconversion between two assemblies. In the self-inactivated homotetramer, KLHDC2's C-terminal Gly-Ser motif mimics a degron and engages the substrate-binding domain of another protomer. True substrates capture the monomeric CRL2KLHDC2, driving E3 activation by neddylation and subsequent substrate ubiquitylation. Non-substrates such as NEDD8 bind KLHDC2 with high affinity, but its slow on rate prevents productive association with CRL2KLHDC2. Without substrate, neddylated CRL2KLHDC2 assemblies are deactivated via distinct mechanisms: the monomer by deneddylation and the tetramer by auto-ubiquitylation. Thus, substrate specificity is amplified by KLHDC2 self-assembly acting like a molecular timer, where only bona fide substrates may bind before E3 ligase inactivation.

BCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric "BH3-in-groove" triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.

Histone lysine demethylases (KDMs) play critical roles in oncogenesis and therefore may be effective targets for anticancer therapy. Using a time-resolved fluorescence resonance energy transfer demethylation screen assay, in combination with multiple orthogonal validation approaches, we identified geldanamycin and its analog 17-DMAG as KDM inhibitors. In addition, we found that these Hsp90 inhibitors increase degradation of the alveolar rhabdomyosarcoma (aRMS) driver oncoprotein PAX3-FOXO1 and induce the repressive epigenetic mark H3K9me3 and H3K36me3 at genomic loci of PAX3-FOXO1 targets. We found that as monotherapy 17-DMAG significantly inhibits expression of PAX3-FOXO1 target genes and multiple oncogenic pathways, induces a muscle differentiation signature, delays tumor growth and extends survival in aRMS xenograft mouse models. The combination of 17-DMAG with conventional chemotherapy significantly enhances therapeutic efficacy, indicating that targeting KDM in combination with chemotherapy may serve as a therapeutic approach to PAX3-FOXO1-positive aRMS.

Phase separation compartmentalizes many cellular pathways. Given that the same interactions that drive phase separation mediate the formation of soluble complexes below the saturation concentration, the contribution of condensates versus complexes to function is sometimes unclear. Here, we characterized several new cancer-associated mutations of the tumor suppressor speckle-type POZ protein (SPOP), a substrate recognition subunit of the Cullin3-RING ubiquitin ligase. This pointed to a strategy for generating separation-of-function mutations. SPOP self-associates into linear oligomers and interacts with multivalent substrates, and this mediates the formation of condensates. These condensates bear the hallmarks of enzymatic ubiquitination activity. We characterized the effect of mutations in the dimerization domains of SPOP on its linear oligomerization, binding to its substrate DAXX, and phase separation with DAXX. We showed that the mutations reduce SPOP oligomerization and shift the size distribution of SPOP oligomers to smaller sizes. The mutations therefore reduce the binding affinity to DAXX but unexpectedly enhance the poly-ubiquitination activity of SPOP toward DAXX. Enhanced activity may be explained by enhanced phase separation of DAXX with the SPOP mutants. Our results provide a comparative assessment of the functional role of complexes versus condensates and support a model in which phase separation is an important factor in SPOP function. Our findings also suggest that tuning of linear SPOP self-association could be used by the cell to modulate activity and provide insights into the mechanisms underlying hypermorphic SPOP mutations. The characteristics of cancer-associated SPOP mutations suggest a route for designing separation-of-function mutations in other phase-separating systems.