TEAD (transcriptional enhanced associate domain) transcription factors (TEAD1-4) serve as the primary effectors of the Hippo signaling pathway in various cancers. Targeted therapy leads to the emergence of resistance and the underlying mechanism of resistance to TEAD inhibition in cancers is less characterized. We uncover that upregulation of the AP-1 (activator protein-1) transcription factors, along with restored YAP (yes-associated protein) and TEAD activity, drives resistance to GNE-7883, a pan-TEAD inhibitor. Acute GNE-7883 treatment abrogates YAP-TEAD binding and attenuates FOSL1 (FOS like 1) activity. TEAD inhibitor resistant cells restore YAP and TEAD chromatin occupancy, acquire additional FOSL1 binding and exhibit increased MAPK (mitogen-activated protein kinase) pathway activity. FOSL1 is required for the chromatin binding of YAP and TEAD. This study describes a clinically relevant interplay between the Hippo and MAPK pathway and highlights the key role of MAPK pathway inhibitors in mitigating resistance to TEAD inhibition in Hippo pathway dependent cancers.

We describe a process for rapid antibody affinity optimization by repertoire mining to identify clones across B cell clonal lineages based on convergent immune responses where antigen-specific clones with the same heavy (VH) and light chain germline segment pairs, or parallel lineages, bind a single epitope on the antigen. We use this convergence framework to mine unique and distinct VH lineages from rat anti-triggering receptor on myeloid cells 2 (TREM2) antibody repertoire datasets with high diversity in the third complementarity-determining loop region (CDR H3) to further affinity-optimize a high-affinity agonistic anti-TREM2 antibody while retaining critical functional properties. Structural analyses confirm a nearly identical binding mode of anti-TREM2 variants with subtle but significant structural differences in the binding interface. Parallel lineage repertoire mining is uniquely tailored to rationally explore the large CDR H3 sequence space in antibody repertoires and can be easily and generally applied to antibodies discovered in vivo.

We were attracted to the therapeutic potential of inhibiting Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING E3 ligase that plays a critical role in regulating the activation of T cells. However, given that only protein-protein interactions were involved, it was unclear whether inhibition by a small molecule would be a viable approach. After screening an ∼6 billion member DNA-encoded library (DEL) using activated Cbl-b, we identified compound 1 as a hit for which the cis-isomer (2) was confirmed by biochemical and surface plasmon resonance (SPR) assays. Our hit optimization effort was greatly accelerated when we obtained a cocrystal structure of 2 with Cbl-b, which demonstrated induced binding at the substrate binding site, namely, the Src homology-2 (SH2) domain. This was quite noteworthy given that there are few reports of small molecule inhibitors that bind to SH2 domains and block protein-protein interactions. Structure- and property-guided optimization led to compound 27, which demonstrated measurable cell activity, albeit only at high concentrations.

The Hippo pathway is among the most frequently altered key signaling pathways in cancer. TEAD1-4 are essential transcription factors and key downstream effectors in the Hippo pathway. Here we identified RNF146 as a ubiquitin ligase (E3) that can catalyze TEAD ubiquitination and negatively regulate their function in cells. We show that this ubiquitin of TEADs is governed by their PARylation state and validated the genetic interaction between RNF146 and the Hippo pathway in cancer cell lines and the model organism Drosophila melanogaster. Furthermore, we demonstrate that pharmacologically induced ubiquitination of TEADs by heterobifunctional chemical inducers of protein degradation (CIDE) molecules can promote potent pan-TEAD degradation. These TEAD-CIDEs can effectively suppress activation of TEAD target genes in a dose-dependent manner and exhibited significant anti-proliferative effects in Hippo-dependent tumor cells, thus phenocopy the effect of genetic ablation of TEAD protein. Collectively, this study demonstrates a post-translational mechanism of TEAD protein regulation and provides a proof-of-concept demonstration that pharmacological induced TEAD ubiquitination could be an effective therapeutic strategy to target Hippo-driven cancers.

The drug discovery landscape is ever‐evolving and constantly demands revolutionary technology advancements in protein expression and production laboratories. We have built a higher‐throughput mid‐scale semi‐automated protein expression and screening platform to accelerate drug discovery research. The workflow described here enables comprehensive expression and purification screening assessment of challenging or difficult‐to‐express recombinant proteins in a fast and efficient manner by delivering small but sufficient amounts of high‐quality proteins. The platform has been implemented for a wide range of applications that include identification of optimal constructs and chaperones for poorly expressing proteins, assessment of co‐expression partners for expressing stable multiprotein complexes, and suitable buffer/additive screening for insoluble or aggregation‐prone proteins. The approach allows parallel expression, purification, and characterization of 24 different samples using co‐infection or a polycistronic approach in insect cells and enables parallel testing of multiple parameters to improve protein yields. The strategy has been successfully adopted for screening intracellular and secreted proteins in Escherichia coli, mammalian transient expression, and baculovirus expression vector systems. Proteins purified from this platform are used for several structural and functional screens, such as negative staining, biochemical activity assays, mass spectrometry, surface plasmon resonance, and DNA‐encoded chemical library screens. In this article, for simplicity, we have focused on detailed expression and purification screening of intracellular protein complexes from insect cells. © 2023 Wiley Periodicals LLC.

Inhibition of NF-κB inducing kinase (NIK) has been pursued as a promising therapeutic target for autoimmune disorders due to its highly regulated role in key steps of the NF-κB signaling pathway. Previously reported NIK inhibitors from our group were shown to be potent, selective, and efficacious, but had higher human dose projections than desirable for immunology indications. Herein we report the clearance-driven optimization of a NIK inhibitor guided by metabolite identification studies and structure-based drug design. This led to the identification of an azabicyclo[3.1.0]hexanone motif that attenuated in vitro and in vivo clearance while maintaining NIK potency and increasing selectivity over other kinases, resulting in a greater than ten-fold reduction in predicted human dose.

An efficient total chemical synthesis of site-specifically sumoylated histone H4 was undertaken to generate homogenously modified mononucleosomes. These were tested as substrates in biochemical assays with the histone H2B-specific ubiquitin ligases Rad6 and Bre1, which revealed the strong inhibition of H2B ubiquitylation by SUMO. This novel negative biochemical crosstalk between SUMO and ubiquitin was also confirmed to exist in human cells.

The post-translational modification of histones by the small ubiquitin-like modifier (SUMO) protein has been associated with gene regulation, centromeric localization, and double-strand break repair in eukaryotes. Although sumoylation of histone H4 was specifically associated with gene repression, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using an H4 chemically modified at Lys12 by SUMO-3 (H4K12su) and incorporated into mononucleosomes and chromatinized plasmids for functional studies. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS (complex of proteins associated with Set1) histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed that H4K12su inhibits both H4 tail acetylation and RNA polymerase II-mediated transcription. Finally, cell-based assays with a SUMO-H4 fusion that mimics H4 tail sumoylation confirmed the negative crosstalk between histone sumoylation and acetylation/methylation. Thus, our studies establish the key role for histone sumoylation in gene silencing and its negative biochemical crosstalk with active transcription-associated marks in human cells.

Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.

The COMPASS complex represents the prototype of the SET1/MLL family of methyltransferases that controls gene transcription by H3K4 methylation (H3K4me). Although H2B monoubiquitination (H2Bub) is well-known as a prerequisite histone mark for COMPASS activity, how the H2Bub-H3K4me crosstalk is catalyzed by COMPASS remains unclear. Here, we report the cryo-EM structures of an extended COMPASS catalytic module (CM) bound to the H2Bub and free nucleosome. The COMPASS CM clamps onto the nucleosome disk-face via an extensive interface to capture the flexible H3 N-terminal tail. The interface also sandwiches a critical Set1 arginine-rich motif (ARM) that auto-inhibits COMPASS. Unexpectedly, without enhancing COMPASS-nucleosome interaction, H2Bub activates the enzymatic assembly by packing against Swd1 and alleviating the inhibitory effect of the Set1 ARM upon fastening it to the acidic patch. By unmasking the spatial configuration of the COMPASS-H2Bub-nucleosome assembly, our studies establish the structural framework for understanding the long-studied H2Bub-H3K4me histone modification crosstalk.

Many G protein-coupled receptors (GPCRs) are organized as dynamic macromolecular complexes in human cells. Unraveling the structural determinants of unique GPCR complexes may identify unique protein:protein interfaces to be exploited for drug development. We previously reported α 1D -adrenergic receptors (α 1D -ARs) – key regulators of cardiovascular and central nervous system function – form homodimeric, modular PDZ protein complexes with cell-type specificity. Towards mapping α 1D -AR complex architecture, biolayer interferometry (BLI) revealed the α 1D -AR C-terminal PDZ ligand selectively binds the PDZ protein scribble (SCRIB) with >8x higher affinity than known interactors syntrophin, CASK and DLG1. Complementary in situ and in vitro assays revealed SCRIB PDZ domains 1 and 4 to be high affinity α 1D -AR PDZ ligand interaction sites. SNAP-GST pull-down assays demonstrate SCRIB binds multiple α 1D -AR PDZ ligands via a co-operative mechanism. Structure-function analyses pinpoint R1110 PDZ4 as a unique, critical residue dictating SCRIB:α 1D -AR binding specificity. The crystal structure of SCRIB PDZ4 R1110G predicts spatial shifts in the SCRIB PDZ4 carboxylate binding loop dictate α 1D -AR binding specificity. Thus, the findings herein identify SCRIB PDZ domains 1 and 4 as high affinity α 1D -AR interaction sites, and potential drug targets to treat diseases associated with aberrant α 1D -AR signaling.

Circadian rhythms are generated by a transcription-based feedback loop where CLOCK:BMAL1 drive transcription of their repressors (PER1/2, CRY1/2), which bind to CLOCK:BMAL1 to close the feedback loop with ~24-hour periodicity. Here we identify a key biochemical and structural difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. While both cryptochromes bind the BMAL1 transactivation domain with similar affinity to sequester it from coactivators, CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We identify a dynamic loop in the secondary pocket that regulates differential binding of cryptochromes to the PAS domain core. Notably, PER2 binding remodels this loop in CRY2 to enhance its affinity for CLOCK:BMAL1, explaining why CRY2 forms an obligate heterodimer with PER2, while CRY1 is capable of repressing CLOCK:BMAL1 both with and without PER2.

The SET1/MLL family of histone methyltransferases is conserved in eukaryotes and regulates transcription by catalyzing histone H3K4 mono-, di-, and tri-methylation. These enzymes form a common five-subunit catalytic core whose assembly is critical for their basal and regulated enzymatic activities through unknown mechanisms. Here, we present the crystal structure of the intact yeast COMPASS histone methyltransferase catalytic module consisting of Swd1, Swd3, Bre2, Sdc1, and Set1. The complex is organized by Swd1, whose conserved C-terminal tail not only nucleates Swd3 and a Bre2-Sdc1 subcomplex, but also joins Set1 to construct a regulatory pocket next to the catalytic site. This inter-subunit pocket is targeted by a previously unrecognized enzyme-modulating motif in Swd3 and features a doorstop-style mechanism dictating substrate selectivity among SET1/MLL family members. By spatially mapping the functional components of COMPASS, our results provide a structural framework for understanding the multifaceted functions and regulation of the H3K4 methyltransferase family.

Cleavage stimulation factor (CstF) is a highly conserved protein complex composed of three subunits that recognizes G/U-rich sequences downstream of the polyadenylation signal of eukaryotic mRNAs. While CstF has been identified over 25 years ago, the architecture and contribution of each subunit to RNA recognition have not been fully understood. In this study, we provide a structural basis for the recruitment of CstF-50 to CstF via interaction with CstF-77 and establish that the hexameric assembly of CstF creates a high affinity platform to target various G/Urich sequences. We further demonstrate that CstF-77 boosts the affinity of the CstF-64 RRM to the RNA targets and CstF-50 fine tunes the ability of the complex to recognize G/U sequences of certain lengths and content.

3'-End processing of pre-mRNAs prior to packaging and export to the cytoplasm of the mature transcript is a highly regulated process executed by several tens of protein factors that recognize poorly conserved RNA signals. Among them is Pcf11, a highly conserved, multidomain protein that links transcriptional elongation, 3'-end processing, and transcription termination. Here we report the structure and biochemical function of Pcf11's C-terminal domain, which is conserved from yeast to humans. We identify a novel zinc-finger fold, resembling a trillium flower. Structural, biochemical, and genetic analyses reveal a highly conserved surface that plays a critical role in both cleavage and polyadenylation. These findings provide further insight into this important protein and its multiple functional roles during cotranscriptional RNA processing.

The phosphorylation state of heptapeptide repeats within the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (PolII) controls the transcription cycle and is maintained by the competing action of kinases and phosphatases. Rtr1 was recently proposed to be the enzyme responsible for the transition of PolII into the elongation and termination phases of transcription by removing the phosphate marker on serine 5, but this attribution was questioned by the apparent lack of enzymatic activity. Here we demonstrate that Rtr1 is a phosphatase of new structure that is auto-inhibited by its own C-terminus. The enzymatic activity of the protein in vitro is functionally important in vivo as well: a single amino acid mutation that reduces activity leads to the same phenotype in vivo as deletion of the protein-coding gene from yeast. Surprisingly, Rtr1 dephosphorylates not only serine 5 on the CTD but also the newly described anti-termination tyrosine 1 marker, supporting the hypothesis that Rtr1 and its homologs promote the transition from transcription to termination.

The phosphatase Rtr1 has been implicated in dephosphorylation of the RNA Polymerase II (RNAPII) C-terminal domain (CTD) during transcription elongation and in regulation of nuclear import of RNAPII. Although it has been shown that Rtr1 interacts with RNAPII in yeast and humans, the specific mechanisms that underlie Rtr1 recruitment to RNAPII have not been elucidated. To address this, we have performed an in-depth proteomic analysis of Rtr1 interacting proteins in yeast. Our studies revealed that hyperphosphorylated RNAPII is the primary interacting partner for Rtr1. To extend these findings, we performed quantitative proteomic analyses of Rtr1 interactions in yeast strains deleted for CTK1, the gene encoding the catalytic subunit of the CTD kinase I (CTDK-I) complex. Interestingly, we found that the interaction between Rtr1 and RNAPII is decreased in ctk1Δ strains. We hypothesize that serine-2 CTD phosphorylation is required for Rtr1 recruitment to RNAPII during transcription elongation.

N-methyl-D-aspartate (NMDA) receptors are calcium-permeable ion channels assembled from four subunits that each have a common membrane topology. The intracellular carboxyl terminal domain (CTD) of each subunit varies in length, is least conserved between subunits, and binds multiple intracellular proteins. We defined a region of interest in the GluN2A CTD, downstream of well-characterized membrane-proximal motifs, that shares only 29% sequence similarity with the equivalent region of GluN2B. GluN2A (amino acids 875-1029) was fused to GST and used as a bait to identify proteins from mouse brain with the potential to bind GluN2A as a function of calcium. Using mass spectrometry we identified calmodulin as a calcium-dependent GluN2A binding partner. Equilibrium fluorescence spectroscopy experiments indicate that Ca2+/calmodulin binds GluN2A with high affinity (5.2 +/- 2.4 nM) in vitro. Direct interaction of Ca2+/calmodulin with GluN2A was not affected by disruption of classic sequence motifs associated with Ca2+/calmodulin target recognition, but was critically dependent upon Trp-1014. These findings provide new insight into the potential of Ca2+/calmodulin, previously considered a GluN1-binding partner, to influence NMDA receptors by direct association. (C) 2014 Elsevier Inc. All rights reserved.