The ligand dependent transcription factor estrogen receptor ERa is a key driver of and important drug target in breast cancer. Patients with advanced disease are typically treated with selective ERa degraders (SERDs), whose therapeutic activity is commonly attributed to induced ERa protein degradation. Yet the exact mechanism and relevance of degradation for clinical efficacy remain unclear. We show that SERDs directly induce ERa SUMOylation, thereby triggering degradation via SUMO-targeted ubiquitin ligases (STUbLs). Inactivation of STUbLs prevents ERa degradation and counterintuitively further sensitizes breast cancer cells to SERDs, rather than conferring resistance. SERD efficacy is independent of ERa degradation, challenging the degradation-centric model of SERD action. Instead, SUMOylation recruits transcriptional co-repressors, turning SUMOylated ERa into a dominant-negative repressor. Thus, SUMOylation rather than degradation is the direct consequence and driver of SERD activity. Our findings position SUMO-inducing drugs as a hitherto underappreciated yet clinically validated therapeutic modality with broad applicability.

Molecular glue degraders (MGDs), such as pomalidomide, induce degradation of non-native substrates by the cullin-RING E3 ligase 4 (CRL4) through its substrate receptor cereblon (CRBN). Here, to explore CRBN programmability, we tested whether reported CRBN-MGD substrates are part of a network of latent CRBN interactors, proteins capable of MGD-induced CRBN binding without detectable degradation. Leveraging a highly parallel protein complementation assay (GluePCA) to measure MGD-induced interaction between CRBN and zinc fingers, we identified ~210 zinc fingers bound to CRBN-pomalidomide, where top binders are already reported as degraded by dedicated MGDs. To map latent CRBN-MGD interactions proteome-wide and define the accessible CRBN interaction space, we combined artificial intelligence-derived protein surface queries (MaSIF-mimicry) with GluePCA. This pipeline identified 6 known and 43 novel CRBN-pomalidomide binders, including orthogonally validated hits. We find that these binders provide privileged starting points for MGD development. We expect this binding-focused workflow to be applicable to other MGD-E3 ligase systems, potentially extending the scope of this emerging drug class.

Chromatin remodeling complexes mobilize nucleosomes and promote transcription factor (TF) binding. Using ensemble and single-molecule assays combined with cryo-electron microscopy (cryo-EM), we studied the interaction between pioneer TFs OCT4-SOX2 and the human BRG1/BRM-associated factor (BAF) complex on nucleosomes. BAF engages TF-bound substrates in two orientations, placing OCT4-SOX2 at either the remodeler ENTRY or EXIT site. At the ENTRY site, OCT4-SOX2 initially coexists with BAF without structural interference. However, continued DNA translocation is expected to cause collisions with bound TFs, which can trigger remodeling direction reversals or may induce TF dissociation. To accommodate TFs at the EXIT site, BAF undergoes structural rearrangements, and ensemble assays reveal a nucleosome subpopulation translocating away from TF-binding sites. Moreover, single-molecule experiments show that nucleosome-bound BAF frequently changes remodeling direction, and we identify an ADP-bound remodeler conformation as a potential intermediate. Together, these findings reveal key aspects of the conformational dynamics and remodeling outcomes underlying BAF processing of TF-bound nucleosomes.

TRIpartite Motif-containing 72 (TRIM72, also known as MG53), a RING-type E3 ubiquitin ligase, is critical for plasma membrane repair. Like other TRIM family proteins, TRIM72 has a conserved architecture comprising RING, B-box, coiled-coil, and C-terminal PRY-SPRY domains. While the coiled-coil domain mediates homo-oligomerization, its specific contribution to the membrane repair machinery remains unclear. In this study, we characterized the structural and dynamic properties of the TRIM72 coiled-coil domain, aiming to elucidate its contribution to membrane association. Small-angle X-ray scattering and molecular dynamics simulations revealed that the coiled-coil domain exhibits significant flexibility, including directional movements perpendicular to the membrane. Cryo-electron microscopy further demonstrated that coiled-coil-mediated oligomerization facilitated the tethering of adjacent liposomes. These findings highlight the role of the coiled-coil domain in supporting higher-order assembly on membranes, providing mechanistic insights into the TRIM72-mediated membrane repair.

Molecular glue degraders (MGDs) are a transformative modality in drug discovery. MGDs that work in concert with the E3 ligase CRL4CRBN can degrade a range of substrates through tailored MGDs. To explore CRL4CRBN reprogrammability, we tested whether reported CRBN-MGD substrates are part of a network of latent CRBN interactors, detectable with generic CRBN-MGDs. Leveraging highly parallel interaction measurement (GluePCA) between CRL4CRBN and human zinc-fingers (ZFs), we identified ~210 ZFs bound to CRBN-pomalidomide, where top binders are already reported as degraded by dedicated MGDs. To map latent CRBN-MGDs interactions proteome-wide, and thus define the immediately accessible CRBN target space, we combined AI-derived protein surface queries (MaSIF-mimicry) with GluePCA. This pipeline identified 6 known and 43 novel CRBN-pomalidomide binders, thereby providing privileged starting points for MGD development. We expect this binding-focused, highly parallel workflow to be readily applicable to other MGD/E3 ligase systems, extending the target landscape of this emerging drug class. ### Competing Interest Statement N.H.T. is a founder and shareholder of Zenith Therapeutics as well as a consultant to Ridgeline Discovery. The laboratory receives financial support from AstraZeneca, Merck KGaA and the Novartis Foundation.

The basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) transcription factor CLOCK:BMAL1 interacts with E-box motifs in the context of nucleosomes to elicit a circadian pattern of gene expression that oscillates with approximately 24 hour periodicity. Core clock genes and other highly rhythmic targets of CLOCK:BMAL1 typically possess a tandem arrangement of E-boxes that is required for robust oscillations. Here, we show that the presence of tandem E-boxes enables CLOCK:BMAL1 to bind more internal sites on the nucleosome, leading to release of DNA from the histone core and the presentation of multiple coactivator binding motifs in close proximity to facilitate multivalent interactions with the coactivator CBP/p300. We show that the transactivation domain (TAD) of BMAL1, essential for CLOCK:BMAL1 activity, interacts with several modular domains of CBP. Deletion of these CBP domains or chemical inhibition of protein-protein interactions with CBP significantly reduces or eliminates CLOCK:BMAL1-driven activity. Altogether, this suggests that multivalent interactions with CBP may play a role in the ability of tandem CLOCK:BMAL1 heterodimers to recruit this limiting cofactor in cells.

Pioneer transcription factors (TFs) engage chromatinized DNA motifs. However, it is unclear how the resultant TF-nucleosome complexes are decoded by co-factors. In humans, the TF p53 regulates cell-cycle progression, apoptosis, and the DNA damage response, with a large fraction of p53-bound sites residing in nucleosome-harboring inaccessible chromatin. We examined the interaction of chromatin-bound p53 with co-factors belonging to the ubiquitin proteasome system (UPS). At two distinct motif locations on the nucleosome (super-helical location [SHL]-5.7 and SHL+5.9), the E3 ubiquitin ligase E6-E6AP was unable to bind nucleosome-engaged p53. The deubiquitinase USP7, on the other hand, readily engages nucleosome-bound p53 in vitro and in cells. A corresponding cryo-electron microscopy (cryo-EM) structure shows USP7 engaged with p53 and nucleosomes. Our work illustrates how chromatin imposes a co-factor-selective barrier for p53 interactors, whereby flexibly tethered interaction domains of co-factors and TFs govern compatibility between co-factors, TFs, and chromatin.

Retrotransposon control in mammals is an intricate process that is effectuated by a broad network of chromatin regulatory pathways. We previously discovered ChAHP, a protein complex with repressive activity against short interspersed element (SINE) retrotransposons that is composed of the transcription factor ADNP, chromatin remodeler CHD4, and HP1 proteins. Here we identify ChAHP2, a protein complex homologous to ChAHP, in which ADNP is replaced by ADNP2. ChAHP2 is predominantly targeted to endogenous retroviruses (ERVs) and long interspersed elements (LINEs) via HP1β-mediated binding of H3K9 trimethylated histones. We further demonstrate that ChAHP also binds these elements in a manner mechanistically equivalent to that of ChAHP2 and distinct from DNA sequence-specific recruitment at SINEs. Genetic ablation of ADNP2 alleviates ERV and LINE1 repression, which is synthetically exacerbated by additional depletion of ADNP. Together, our results reveal that the ChAHP and ChAHP2 complexes function to control both nonautonomous and autonomous retrotransposons by complementary activities, further adding to the complexity of mammalian transposon control.

The encoding and evolution of specificity and affinity in protein-protein interactions is poorly understood. Here, we address this question by quantifying how all mutations in one protein, JUN, alter binding to all other members of a protein family, the 54 human basic leucine zipper transcription factors. We fit a global thermodynamic model to the data to reveal that most affinity changing mutations equally affect JUN’s affinity to all its interaction partners. Mutations that alter binding specificity are relatively rare but distributed throughout the interaction interface. Specificity is determined both by features that promote on-target interactions and by those that prevent off-target interactions. Approximately half of the specificity-defining residues in JUN contribute both to promoting on-target binding and preventing off-target binding. Nearly all specificity-altering mutations in the interaction interface are pleiotropic, also altering affinity to all partners. In contrast, mutations outside the interface can tune global affinity without affecting specificity. Our results reveal the distributed encoding of specificity and affinity in an interaction interface and how coiled-coils provide an elegant solution to the challenge of optimizing both specificity and affinity in a large protein family. Here, the authors build a map of how mutations alter the binding of JUN to all 54 human bZIP proteins, revealing how affinity and specificity can be tuned and how determinants of specificity distribute in a protein interaction interface.

Substrate specificity determines protease functions in physiology and in clinical and biotechnological applications, yet quantitative cleavage information is often unavailable, biased, or limited to a small number of events. Here, we develop qPISA (quantitative Protease specificity Inference from Substrate Analysis) to study Dipeptidyl Peptidase Four (DPP4), a key regulator of blood glucose levels. We use mass spectrometry to quantify >40,000 peptides from a complex, commercially available peptide mixture. By analyzing changes in substrate levels quantitatively instead of focusing on qualitative product identification through a binary classifier, we can reveal cooperative interactions within DPP4's active pocket and derive a sequence motif that predicts activity quantitatively. qPISA distinguishes DPP4 from the related C. elegans DPF-3 (a DPP8/9-orthologue), and we relate the differences to the structural features of the two enzymes. We demonstrate that qPISA can direct protein engineering efforts like the stabilization of GLP-1, a key DPP4 substrate used in the treatment of diabetes and obesity. Thus, qPISA offers a versatile approach for profiling protease and especially exopeptidase specificity, facilitating insight into enzyme mechanisms and biotechnological and clinical applications.

DNA origami, a method for constructing nanostructures from DNA, offers potential for diverse scientific and technological applications due to its ability to integrate various molecular functionalities in a programmable manner. In this study, we examined the impact of internal crossover distribution and the compositional uniformity of staple strands on the structure of multilayer DNA origami using cryogenic electron microscopy (cryo-EM) single-particle analysis. A refined DNA object was utilized as an alignment framework in a host-guest model, where we successfully resolved an 8 kDa thrombin binding aptamer (TBA) linked to the host object. Our results broaden the spectrum of DNA in structural applications.

Assembly Quality Control (AQC) E3 ubiquitin ligases target incomplete or incorrectly assembled protein complexes for degradation. The CUL4‐RBX1‐DDB1‐DCAF12 (CRL4 DCAF12 ) E3 ligase preferentially ubiquitinates proteins that carry a C‐terminal double glutamate (di‐Glu) motif. Reported CRL4 DCAF12 di‐Glu‐containing substrates include CCT5, a subunit of the TRiC chaperonin. How DCAF12 engages its substrates and the functional relationship between CRL4 DCAF12 and CCT5/TRiC is currently unknown. Here, we present the cryo‐EM structure of the DDB1‐DCAF12‐CCT5 complex at 2.8 Å resolution. DCAF12 serves as a canonical WD40 DCAF substrate receptor and uses a positively charged pocket at the center of the β‐propeller to bind the C‐terminus of CCT5. DCAF12 specifically reads out the CCT5 di‐Glu side chains, and contacts other visible degron amino acids through Van der Waals interactions. The CCT5 C‐terminus is inaccessible in an assembled TRiC complex, and functional assays demonstrate that DCAF12 binds and ubiquitinates monomeric CCT5, but not CCT5 assembled into TRiC. Our biochemical and structural results suggest a previously unknown role for the CRL4 DCAF12 E3 ligase in overseeing the assembly of a key cellular complex.

Proteins function in crowded cellular environments in which they must bind to specific target proteins but also avoid binding to many other off-target proteins. In large protein families this task is particularly challenging because many off-target proteins have very similar structures. How this specificity of physical protein-protein interactions in cellular networks is encoded and evolves is not very well understood. Here we address the question of specificity-encoding by comprehensively quantifying the effects of all mutations in one protein, JUN, on its binding to all other members of a protein family, the 54 human basic leucine zipper transcription factors. Fitting a global thermodynamic model to the data reveals that most affinity changing mutations equally affect JUN’s propensity to bind to all its interaction partners. Mutations that alter the specificity of binding are much rarer. These specificity-altering mutations are, however, distributed throughout the JUN interaction interface. JUN’s interaction specificity is encoded by both positive determinants that promote on-target interactions and negative determinants that prevent off-target interactions. Indeed, about half of the specificity-defining residues in JUN have dual functions and both promote on-target binding and prevent off-target binding. Whereas nearly all mutations that alter specificity are pleiotropic and also alter the affinity of binding to all interaction partners, the converse is not true with mutations outside of the interface able to tune affinity without affecting specificity. Our results provide the first global view of how mutations in a protein affect binding to all its potential interaction partners and reveal the distributed encoding of specificity and affinity in an interaction interface. They also show how the modular architecture of coiled-coils provides an elegant solution to the challenge of optimising specificity and affinity in a large protein family.

GUIFold is a graphical user interface for the open-source structure prediction pipeline AlphaFold2. A particular emphasis lies on tracking prediction jobs in a database as well as user-friendly submission to queueing systems. GUIFold is built on top of a modified AlphaFold2 pipeline that primarily allows more control of the feature generation step. Additionally, the application provides an evaluation pipeline to rank predictions and visualize confidence metrics. GUIFold can be installed along with AlphaFold2 in a virtual environment and, after initial setup, allows running jobs without particular technical expertise.

The E3-ubiquitin ligase E6AP degrades p53 when complexed with the viral protein E6 from human papilloma virus (HPV), which contributes to the transformation of cells in HPV-related cancers. Previous crystal structures of the E6AP-E6-p53 ternary complex have implicated a peptide containing an LxxLL motif from E6AP as the interface between the three proteins. However, the contributions to the ternary complex from the remainder of the E6AP protein remain unknown. We reexamined this complex using cryo-EM and full-length proteins and find additional protein interaction interfaces involving a previously uncharacterized domain of E6AP. Additionally, we observe that the ternary complex forms both 1:1:1 and 2:2:2 stochiometric complexes comprised of E6AP, E6 and p53.

The basic helix–loop–helix (bHLH) family of transcription factors recognizes DNA motifs known as E-boxes (CANNTG) and includes 108 members 1 . Here we investigate how chromatinized E-boxes are engaged by two structurally diverse bHLH proteins: the proto-oncogene MYC-MAX and the circadian transcription factor CLOCK-BMAL1 (refs. 2 , 3 ). Both transcription factors bind to E-boxes preferentially near the nucleosomal entry–exit sites. Structural studies with engineered or native nucleosome sequences show that MYC-MAX or CLOCK-BMAL1 triggers the release of DNA from histones to gain access. Atop the H2A–H2B acidic patch 4 , the CLOCK-BMAL1 Per-Arnt-Sim (PAS) dimerization domains engage the histone octamer disc. Binding of tandem E-boxes 5 – 7 at endogenous DNA sequences occurs through direct interactions between two CLOCK-BMAL1 protomers and histones and is important for circadian cycling. At internal E-boxes, the MYC-MAX leucine zipper can also interact with histones H2B and H3, and its binding is indirectly enhanced by OCT4 elsewhere on the nucleosome. The nucleosomal E-box position and the type of bHLH dimerization domain jointly determine the histone contact, the affinity and the degree of competition and cooperativity with other nucleosome-bound factors.

Histone deacetylase 6 (HDAC6) is an atypical lysine deacetylase with tandem catalytic domains and an ubiquitin-binding zinc finger domain. HDAC6 is involved in various biological processes, such as cell motility or stress responses, and has been implicated in pathologies ranging from cancer to neurodegeneration. Due to this broad range of functions, there has been considerable interest in developing HDAC6-specific small molecule inhibitors, several of which are already available. The crystal structure of the tandem catalytic domains of zebrafish HDAC6 has revealed an arrangement with twofold symmetry and extensive surface interaction between the catalytic domains. Further dissection of the biochemical properties of HDAC6 and the development of novel inhibitors will benefit from being able to routinely express high-quality protein. We present here our optimized protocol for expression and crystallization of the zebrafish tandem catalytic domains.

Molecular glue degraders are an effective therapeutic modality, but their design principles are not well understood. Recently, several unexpectedly diverse compounds were reported to deplete cyclin K by linking CDK12–cyclin K to the DDB1–CUL4–RBX1 E3 ligase. Here, to investigate how chemically dissimilar small molecules trigger cyclin K degradation, we evaluated 91 candidate degraders in structural, biophysical and cellular studies and reveal all compounds acquire glue activity via simultaneous CDK12 binding and engagement of DDB1 interfacial residues, in particular Arg928. While we identify multiple published kinase inhibitors as cryptic degraders, we also show that these glues do not require pronounced inhibitory properties for activity and that the relative degree of CDK12 inhibition versus cyclin K degradation is tuneable. We further demonstrate cyclin K degraders have transcriptional signatures distinct from CDK12 inhibitors, thereby offering unique therapeutic opportunities. The systematic structure–activity relationship analysis presented herein provides a conceptual framework for rational molecular glue design.