Group III metabotropic glutamate receptors (mGluRs) are critical signaling molecules that regulate strength, homeostasis, and plasticity of glutamatergic synaptic signaling. These receptors are engaged in transsynaptic interactions with extracellular leucine-rich repeat and fibronectin type III domain-containing (ELFN) cell adhesion proteins. ELFN proteins have been shown to play a critical role in regulation of activity and localization of mGluRs activity in vivo, yet the exact nature of their regulatory interaction has remained unknown. Here, we present a cryo-electron microscopy structure of the ELFN-mGluR complex. We identify a specific ELFN-binding pocket on mGluRs involved in its allosteric regulation through the network of residues affecting the orthosteric ligand binding site. We further uncover cooperativity whereby mGluR activation increases their association with ELFN proteins as a potential feedback mechanism to regulate synaptic strength. Last, we determine that disruption in mGluR-ELFN interaction is a recurring mechanism underlying several neurological conditions as we delineate their structure-functional etiology.

Current endocrine therapies for breast cancer are used to treat the 70 percent of estrogen receptor (ER) positive breast cancers, but de novo and acquired resistance drive progressive metastatic disease. Hormone therapy development efforts have used similar chemical targeting strategies such that the structural basis of ligand efficacy is not clear. More than a third of patients who develop endocrine therapy resistance have hotspot constitutively activating mutations in the ligand binding domain, most notably Y537S and D538G. While these mutants have been studied extensively in stabilizing agonist conformation and constitutive activity in the absence of estradiol, the structural basis for why these mutations cause loss of efficacy for antagonists is not as well studied. To improve our understanding of structural mechanisms of therapeutic efficacy in the ESR1 mutant setting, we synthesized a diverse series of compounds including ∼100 ligands based on a high affinity adamantyl scaffold. We diversified the ligand side chains with various pharmacophores to understand the structural basis of ligand potency and efficacy on wild type and mutant ERs. Crystal structures of 20 ligands bound to Erα-LBD revealed how the chemical side chain modulated receptor structure to stabilize different conformational states to drive anti-cancer activity. Previous work revealed how the ER mutants drive constitutive activity by stabilizing the active ER conformation, but this state does not occur with antagonist bound ER. Long timescale molecular dynamics simulations were used to understand conformational changes induced by these mutations in the context of the antagonist conformation of the key helix 12 (h12), which determines pathogenic versus therapeutic activity states. The Y537S mutation stabilized h12 in one of two stable antagonist substates with implications for generating treatment resistance. In contrast, the D538G mutation, which is characterized by a helix-breaking effect, generated multiple metastable conformational substates. These findings support a model where the ligand side chains alter the relative stability of different antagonist substates to drive efficacy, with conformations that are different in the wild type versus mutant ERα bound to endocrine therapies. Date of Presentation October 17, 2024

Talin, a key integrin activator, is essential for cellular adhesion, signal transduction, and mechanical stability. Its transition between autoinhibited and active conformations allows dynamic regulation of adhesion in response to environmental cues. Cholesterol-rich membrane microdomains, such as lipid rafts, organize and stabilize signaling platforms, influencing talin and integrin conformational states. Cholesterol is a switch modulating talin activation, integrin binding, and adhesion. Environmental pollutants, including heavy metals and air toxins, disrupt cholesterol homeostasis, destabilize lipid rafts, and interfere with talin-integrin interactions. These disruptions impair adhesion, tissue repair, and signaling fidelity, contributing to atherosclerosis and cancer metastasis. Understanding talin's interaction with cholesterol-rich domains offers critical insights into adhesion regulation and reveals the broader impact of environmental toxicants on cellular function. This framework emphasizes the importance of membrane composition, particularly cholesterol, in mediating the effects of environmental stressors and suggests potential therapeutic interventions.

Disclosure: J.C. Nwachukwu: None. C.K. Min: None. R.R. Kobylski: None. T.J. Kim: None. Y. Hou: None. S. Kim: None. G.R. Hancock: None. T. Izard: None. S.W. Fanning: None. B.S. Katzenellenbogen: None. J.A. Katzenellenbogen: None. K.W. Nettles: None. Drugs targeting the estrogen receptor-α (ER) fall into two chemical classes, selective estrogen receptor modulators and degraders (SERMs and SERDs), which are effective treatments for hormone-responsive breast cancer. Compounds in these chemical classes typically show similar optimized efficacy in pre-clinical models of breast cancer, including in the context of ER-activating mutations (e.g. ER Y537S) and other resistance pathways, limiting our ability to identify small differences that may be more impactful clinically. To understand structural and molecular drivers of ER antagonism, we expanded the repertoire of ER targeting strategies. We characterized 108 compounds derived from a high-affinity adamantyl scaffold, incorporating 8 distinct classes of pharmacophores, each featuring a diverse array of chemical groups. The chemically diverse ligands revealed that non-canonical targeting approaches produced complex structure activity relationships across a panel of hormone sensitive and resistant breast cancer cell lines and reporter systems, activity that was often more efficacious than existing ER-targeted therapies. We obtained X-ray crystal structures of 25 adamantyl ligands bound to ER resistance mutants. This revealed how the different ligand chemical groups generated diverse structural effects on the surface binding site for transcriptional regulatory proteins that regulate gene expression. Molecular dynamics simulations showed that ER Y537S and D538G stabilized different antagonist conformers, or substates, from the wild type receptor, suggesting a structural basis for resistance to ER antagonists. We selected a subset of 11 adamantyls for a computational approach called “ligand class analysis” (LCA). LCA leverages the wide range of growth inhibitory effects of these related compounds (i.e. a compound class) to provide the statistical robustness for machine learning the underlying mechanisms of action. RNA-seq from MCF-7 cells expressing the ER Y537S resistance allele revealed ∼1600 genes differentially regulated by the 11 adamantyl ligands. We used the growth inhibitory effect of each ligand as the dependent variable in machine learning and identified a gene set response pattern with >95% predictive power for ligand efficacy. Defining causal links from ligand to receptor structure and the cellular mediators of growth inhibition revealed basic principles of allosteric signaling and transcriptional regulation. LCA differentiated these outcome-focused ER signaling pathways from the full set of ligand-regulated cellular effects, while diversification of the ligand-receptor structures identified new ways of modulating ER activity for targeting endocrine resistant breast cancer. Supported by NIH R01CA275142, R01CA220284, R37CA27934, and the Breast Cancer Research Foundation BCRF-083 and BCRF-084 Presentation: Saturday, July 12, 2025

Talin regulates crucial cellular functions, including cell adhesion and motility, and affects human diseases. Triggered by mechanical forces, talin plays crucial roles in facilitating the formation of focal adhesions and recruiting essential focal adhesion regulatory elements such as vinculin. The structural flexibility allows talin to fine-tune its signaling responses. This study presents our 2.7 & Aring; cryoEM structures of talin, which surprisingly uncovers several auto-inhibitory states. Contrary to previous suggestions, our structures reveal that (1) the first and last three domains are not involved in maintaining talin in its closed state and are mobile, (2) the talin F-actin and membrane binding domain are loosely attached and thus available for binding, and (3) the main force-sensing domain is oriented with its vinculin binding sites ready for release. These structural snapshots offer insights and advancements in understanding the dynamic talin activation mechanism, which is crucial for mediating cell adhesion. Talin controls cell adhesion and motility, impacting disease. 2.7 & Aring; cryoEM structures reveal mobile membrane and F-actin binding domains, and force-sensing readiness, providing insights into the dynamic activation of talin in mediating cell adhesion.

The estrogen receptor-α (ER) is thought to function only as a homodimer but responds to a variety of environmental, metazoan, and therapeutic estrogens at subsaturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations—receptor conformational heterodimers—mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining the binding of the same ligand in crystal structures of ER in the agonist vs. antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist vs. antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from the ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric vs. dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing different modes for ligand-dependent regulation of ER activity.

Attachment between cells is crucial for almost all aspects of the life of cells. These inter-cell adhesions are mediated by the binding of transmembrane cadherin receptors of one cell to cadherins of a neighboring cell. Inside the cell, cadherin binds β-catenin, which interacts with α-catenin. The transitioning of cells between migration and adhesion is modulated by α-catenin, which links cell junctions and the plasma membrane to the actin cytoskeleton. At cell junctions, a single β-catenin/α-catenin heterodimer slips along filamentous actin in the direction of cytoskeletal tension which unfolds clustered heterodimers to form catch bonds with F-actin. Outside cell junctions, α-catenin dimerizes and links the plasma membrane to F-actin. Under cytoskeletal tension, α-catenin unfolds and forms an asymmetric catch bond with F-actin. To understand the mechanism of this important α-catenin function, we determined the 2.7 Å cryogenic electron microscopy (cryoEM) structures of filamentous actin alone and bound to human dimeric α-catenin. Our structures provide mechanistic insights into the role of the α-catenin interdomain interactions in directing α-catenin function and suggest a bivalent mechanism. Further, our cryoEM structure of human monomeric α-catenin provides mechanistic insights into α-catenin autoinhibition. Collectively, our structures capture the initial α-catenin interaction with F-actin before the sensing of force, which is a crucial event in cell adhesion and human disease.

Plakophilin-3 is a ubiquitously expressed protein found widely in epithelial cells and is a critical component of desmosomes. The plakophilin-3 carboxy-terminal domain harbors nine armadillo repeat motifs with largely unknown functions. Here, we report the 5 Å cryogenic electron microscopy (cryoEM) structure of the armadillo repeat motif domain of plakophilin-3, one of the smaller cryoEM structures reported to date. We find that this domain is a monomer or homodimer in solution. In addition, using an in vitro actin co-sedimentation assay, we show that the armadillo repeat domain of plakophilin-3 directly interacts with F-actin. This feature, through direct interactions with actin filaments, could be responsible for the observed association of extra-desmosomal plakophilin-3 with the actin cytoskeleton directly attached to the adherens junctions in A431 epithelial cells. Further, we demonstrate, through lipid binding analyses, that plakophilin-3 can effectively be recruited to the plasma membrane through phosphatidylinositol-4,5-bisphosphate-mediated interactions. Collectively, we report on novel properties of plakophilin-3, which may be conserved throughout the plakophilin protein family and may be behind the roles of these proteins in cell–cell adhesion.

The regulation of cell-cell junctions during epidermal morphogenesis ensures tissue integrity, a process regulated by α-catenin. This cytoskeletal protein connects the cadherin complex to filamentous actin at cell-cell junctions. The cadherin-catenin complex plays key roles in cell physiology, organism development, and disease. While mutagenesis of Caenorhabditis elegans cadherin and catenin shows that these proteins are key for embryonic morphogenesis, we know surprisingly little about their structure and attachment to the cytoskeleton. In contrast to mammalian α-catenin that functions as a dimer or monomer, the α-catenin ortholog from C. elegans, HMP1 for humpback, is a monomer. Our cryogenic electron microscopy (cryoEM) structure of HMP1/α-catenin reveals that the amino- and carboxy-terminal domains of HMP1/α-catenin are disordered and not in contact with the remaining HMP1/α-catenin middle domain. Since the carboxy-terminal HMP1/α-catenin domain is the F-actin-binding domain (FABD), this interdomain constellation suggests that HMP1/α-catenin is constitutively active, which we confirm biochemically. Our perhaps most surprising finding, given the high sequence similarity between the mammalian and nematode proteins, is our cryoEM structure of HMP1/α-catenin bound to F-actin. Unlike the structure of mammalian α-catenin bound to F-actin, binding to F-actin seems to allosterically convert a loop region of the HMP1/α-catenin FABD to extend an HMP1/α-catenin FABD α-helix. We use cryoEM and bundling assays to show for the first time how the FABD of HMP1/α-catenin bundles actin in the absence of force. Collectively, our data advance our understanding of α-catenin regulation of cell-cell contacts and additionally aid our understanding of the evolution of multicellularity in metazoans.

G protein-coupled receptors (GPCR) form the largest family of proteins (∼800 GPCRs) encoded in mammalian genomes that detect extracellular signals to program cellular response. They are essential to understanding physiology, disease, and drug development. More than 100 GPCRs are still awaits to identify its endogenous ligand and hence classified as orphan GPCRs. However, these orphan GPCRs have been implicated in several disorders from cancers to neurological diseases. Yet, in many cases, their mechanisms, ligands, and signaling reactions are poorly understood. One such orphan receptor in nervous system is GPR158 that is highly expressed in the brain where it controls synapse formation and function. GPR158 has also been implicated in depression, carcinogenesis, and cognition. However, the structural organization and signaling mechanism of GPR158 are largely unknown. We employed single-particle cryogenic electron microscopy (cryoEM) to obtain structures of GPR158 in the apo state, and in complex with RGS7-Gβ5, a regulator of GPCR signaling. The structure reveals highly unique domain organization of GPR158, not documented previously in any GPCRs structures. The homodimeric organization of protomers, phospholipids interaction and the presence of an extracellular Cache domain, an unusual ligand-binding domain in GPCRs provide insights into the unusual biology of this orphan receptor and the formation of GPCR-RGS complexes. These structures could help to deorphanize the receptor by serving the template for structure-based discovery of its ligands that would leads to exploration of mechanistic of GPR158 mediated neuronal signaling.

Multi-target compounds have become increasingly important for the development of safer and more effective drug candidates. In this work, we devised a combined ligand-based and structure-based multi-target repurposing strategy and applied it to a series of hexahydrocyclopenta[c]quinoline compounds synthesized previously. The in silico analyses identified human Carbonic Anhydrases (hCA) and Estrogen Receptors (ER) as top scoring candidates for dual modulation. hCA isoforms IX and XII, and ER subtypes ER⍺ and/or ERβ are co-expressed in various cancer cell types, including breast and prostate cancer cells. ER⍺ is the primary target of anti-estrogen therapy in breast cancer, and the hCA IX isoform is a therapeutic target in triple-negative breast cancer. ER⍺-mediated transcriptional programs and hCA activity in cancer cells promote favorable microenvironments for cell proliferation. Interestingly, several lines of evidence indicate that the combined modulation of these two targets may provide significant therapeutic benefits. Moving from these first results, two additional hexahydrocyclopenta[c]quinoline derivatives bearing a sulfonamide zinc binding group (hCA) and a phenolic hydroxyl (ER) pharmacophoric group placed at the appropriate locations were designed and synthesized. Interestingly, these compounds were able to directly modulate the activities of both hCA and ER targets. In cell-based assays, they inhibited proliferation of breast and prostate cancer cells with micromolar potency and cell type-selective efficacy. The compounds inhibited hCA activity with nanomolar potency and isoform-selectivity. In transactivation assays, they reduced estrogen-driven ER activity with micro-molar potency. Finally, crystal structures of the synthesized ligands in complex with the two targets revealed that the compounds bind directly to the hCA active site, as well as to the ER ligand-binding domain, providing structural explanation to the observed activity and a rationale for optimization of their dual activity. To the best of our knowledge, this work describes the design, synthesis and biological characterization of the first dual modulators of hCA and ER, laying the ground for the structure-based optimization of their multi-target activity.