UM171 is a potent agonist of ex vivo human haematopoietic stem cell self-renewal¹. By co-opting KBTBD4, a substrate receptor of the CUL3–RING E3 ubiquitin ligase (CRL3) complex, UM171 promotes the degradation of the LSD1–CoREST corepressor complex, thereby limiting haematopoietic stem cell attrition^2,3. However, the direct target and mechanism of action of UM171 remain unclear. Here we show that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1/2 to promote corepressor degradation. Through proteomics and chemical inhibitor studies, we identify the principal target of UM171 as HDAC1/2. Cryo-electron microscopy analysis of dimeric KBTBD4 bound to UM171 and the LSD1–HDAC1–CoREST complex identifies an asymmetric assembly in which a single UM171 molecule enables a pair of KELCH-repeat propeller domains to recruit the HDAC1 catalytic domain. One KBTBD4 propeller partially masks the rim of the HDAC1 active site, which is exploited by UM171 to extend the E3–neosubstrate interface. The other propeller cooperatively strengthens HDAC1 binding through a distinct interface. The overall CoREST–HDAC1/2–KBTBD4 interaction is further buttressed by the endogenous cofactor inositol hexakisphosphate, which acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces is demonstrated by base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, we reveal how the cooperativity offered by a dimeric CRL3 E3 can be leveraged by a small molecule degrader.

Transcriptional regulation lies at the core of the circadian clockwork, but how the clock-related transcription machinery controls the circadian phase is not understood. Here, we show both in human cells and in mice that RuvB-like ATPase 2 (RUVBL2) interacts with other known clock proteins on chromatin to regulate the circadian phase. Pharmacological perturbation of RUVBL2 with the adenosine analog compound cordycepin resulted in a rapid-onset 12-hour clock phase-shift phenotype at human cell, mouse tissue, and whole-animal live imaging levels. Using simple peripheral injection treatment, we found that cordycepin penetrated the blood-brain barrier and caused rapid entrainment of the circadian phase, facilitating reduced duration of recovery in a mouse jet-lag model. We solved a crystal structure for human RUVBL2 in complex with a physiological metabolite of cordycepin, and biochemical assays showed that cordycepin treatment caused disassembly of an interaction between RUVBL2 and the core clock component BMAL1. Moreover, we showed with spike-in ChIP-seq analysis and binding assays that cordycepin treatment caused disassembly of the circadian super-complex, which normally resides at E-box chromatin loci such as PER1, PER2, DBP, and NR1D1. Mathematical modeling supported that the observed type 0 phase shifts resulted from derepression of E-box clock gene transcription.

Cancer mutations can create neomorphic protein–protein interactions to drive aberrant function^1,2. As a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, KBTBD4 is recurrently mutated in medulloblastoma³, the most common embryonal brain tumour in children⁴. These mutations impart gain-of-function to KBTBD4 to induce aberrant degradation of the transcriptional corepressor CoREST⁵. However, their mechanism remains unresolved. Here we establish that KBTBD4 mutations promote CoREST degradation through engaging HDAC1/2 as the direct target of the mutant substrate receptor. Using deep mutational scanning, we chart the mutational landscape of the KBTBD4 cancer hotspot, revealing distinct preferences by which insertions and substitutions can promote gain-of-function and the critical residues involved in the hotspot interaction. Cryo-electron microscopy analysis of two distinct KBTBD4 cancer mutants bound to LSD1–HDAC1–CoREST reveals that a KBTBD4 homodimer asymmetrically engages HDAC1 with two KELCH-repeat β-propeller domains. The interface between HDAC1 and one of the KBTBD4 β-propellers is stabilized by the medulloblastoma mutations, which insert a bulky side chain into the HDAC1 active site pocket. Our structural and mutational analyses inform how this hotspot E3–neosubstrate interface can be chemically modulated. First, we unveil a converging shape-complementarity-based mechanism between gain-of-function E3 mutations and a molecular glue degrader, UM171. Second, we demonstrate that HDAC1/2 inhibitors can block the mutant KBTBD4–HDAC1 interface and proliferation of KBTBD4-mutant medulloblastoma cells. Altogether, our work reveals the structural and mechanistic basis of cancer mutation-driven neomorphic protein–protein interactions.

Inositol hexakisphosphate (IP6) is an endogenous organic molecule present in eukaryotes. First characterized as a phosphorus-storage metabolite, it has been subsequently discovered to play a role in modulating a multitude of different biological pathways. Here, we provide a concise overview of the involvement of IP6 in the ubiquitin-proteasome system (UPS). As an allosteric regulator, a molecular glue, and a potential prosthetic group, IP6 directly impacts the activities of multiple major UPS components. We specifically highlight the structural mechanisms through which IP6 binds to individual proteins or multi-protein complexes to control their functions. The serendipitous discovery of IP6 in various protein structures raises questions about the prevalence, identity, and regulation of soluble inositol polyphosphates in the UPS, which have potential translational implications.

An efficient total chemical synthesis of site-specifically sumoylated histone H4 revealed its negative biochemical crosstalk with histone H2B ubiquitylation in vitro and in human cells.

Cancer mutations can create neomorphic protein-protein interactions to drive aberrant function ¹ . As a substrate receptor of the CULLIN3-RBX1 E3 ubiquitin ligase complex, KBTBD4 is recurrently mutated in medulloblastoma (MB) ² , the most common embryonal brain tumor in children, and pineoblastoma ³ . These mutations impart gain-of-function to KBTBD4 to induce aberrant degradation of the transcriptional corepressor CoREST ⁴ . However, their mechanism of action remains unresolved. Here, we elucidate the mechanistic basis by which KBTBD4 mutations promote CoREST degradation through engaging HDAC1/2, the direct neomorphic target of the substrate receptor. Using deep mutational scanning, we systematically map the mutational landscape of the KBTBD4 cancer hotspot, revealing distinct preferences by which insertions and substitutions can promote gain-of-function and the critical residues involved in the hotspot interaction. Cryo-electron microscopy (cryo-EM) analysis of two distinct KBTBD4 cancer mutants bound to LSD1-HDAC1-CoREST reveals that a KBTBD4 homodimer asymmetrically engages HDAC1 with two KELCH-repeat propeller domains. The interface between HDAC1 and one of the KBTBD4 propellers is stabilized by the MB mutations, which directly insert a bulky side chain into the active site pocket of HDAC1. Our structural and mutational analyses inform how this hotspot E3-neo-substrate interface can be chemically modulated. First, our results unveil a converging shape complementarity-based mechanism between gain-of-function E3 mutations and a molecular glue degrader, UM171. Second, we demonstrate that HDAC1/2 inhibitors can block the mutant KBTBD4-HDAC1 interface, the aberrant degradation of CoREST, and the growth of KBTBD4-mutant MB models. Altogether, our work reveals the structural and mechanistic basis of cancer mutation-driven neomorphic protein-protein interactions and pharmacological strategies to modulate their action for therapeutic applications.