Non-coding 7S RNA inhibits transcription via mitochondrial RNA polymerase dimerization.

The mitochondrial genome encodes 13 components of the oxidative phosphorylation system, and altered mitochondrial transcription drives various human pathologies. A polyadenylated, non-coding RNA molecule known as 7S RNA is transcribed from a region immediately downstream of the light strand promoter in mammalian cells, and its levels change rapidly in response to physiological conditions. Here, we report that 7S RNA has a regulatory function, as it controls levels of mitochondrial transcription both in vitro and in cultured human cells. Using cryo-EM, we show that POLRMT dimerization is induced by interactions with 7S RNA. The resulting POLRMT dimer interface sequesters domains necessary for promoter recognition and unwinding, thereby preventing transcription initiation. We propose that the non-coding 7S RNA molecule is a component of a negative feedback loop that regulates mitochondrial transcription in mammalian cells.

Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies.

Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.

Structural Basis of Functional Transitions in Mammalian NMDA Receptors.

Excitatory neurotransmission meditated by glutamate receptors including N-methyl-D-aspartate receptors (NMDARs) is pivotal to brain development and function. NMDARs are heterotetramers composed of GluN1 and GluN2 subunits, which bind glycine and glutamate, respectively, to activate their ion channels. Despite importance in brain physiology, the precise mechanisms by which activation and inhibition occur via subunit-specific binding of agonists and antagonists remain largely unknown. Here, we show the detailed patterns of conformational changes and inter-subunit and -domain reorientation leading to agonist-gating and subunit-dependent competitive inhibition by providing multiple structures in distinct ligand states at 4 Å or better. The structures reveal that activation and competitive inhibition by both GluN1 and GluN2 antagonists occur by controlling the tension of the linker between the ligand-binding domain and the transmembrane ion channel of the GluN2 subunit. Our results provide detailed mechanistic insights into NMDAR pharmacology, activation, and inhibition, which are fundamental to the brain physiology.

STING Polymer Structure Reveals Mechanisms for Activation, Hyperactivation, and Inhibition.

How the central innate immune protein, STING, is activated by its ligands remains unknown. Here, using structural biology and biochemistry, we report that the metazoan second messenger 2'3'-cGAMP induces closing of the human STING homodimer and release of the STING C-terminal tail, which exposes a polymerization interface on the STING dimer and leads to the formation of disulfide-linked polymers via cysteine residue 148. Disease-causing hyperactive STING mutations either flank C148 and depend on disulfide formation or reside in the C-terminal tail binding site and cause constitutive C-terminal tail release and polymerization. Finally, bacterial cyclic-di-GMP induces an alternative active STING conformation, activates STING in a cooperative manner, and acts as a partial antagonist of 2'3'-cGAMP signaling. Our insights explain the tight control of STING signaling given varying background activation signals and provide a therapeutic hypothesis for autoimmune syndrome treatment.

The Architecture of Talin1 Reveals an Autoinhibition Mechanism.

Focal adhesions (FAs) are protein machineries essential for cell adhesion, migration, and differentiation. Talin is an integrin-activating and tension-sensing FA component directly connecting integrins in the plasma membrane with the actomyosin cytoskeleton. To understand how talin function is regulated, we determined a cryoelectron microscopy (cryo-EM) structure of full-length talin1 revealing a two-way mode of autoinhibition. The actin-binding rod domains fold into a 15-nm globular arrangement that is interlocked by the integrin-binding FERM head. In turn, the rod domains R9 and R12 shield access of the FERM domain to integrin and the phospholipid PIP2 at the membrane. This mechanism likely ensures synchronous inhibition of integrin, membrane, and cytoskeleton binding. We also demonstrate that compacted talin1 reversibly unfolds to an ∼60-nm string-like conformation, revealing interaction sites for vinculin and actin. Our data explain how fast switching between active and inactive conformations of talin could regulate FA turnover, a process critical for cell adhesion and signaling.

Brother's Keeper: Wild-Type Mutant K-Ras Dimers Limit Oncogenesis.

K-Ras is the undisputed champion of oncogenes, yet our ability to interfere with its oncogenic function is hampered by insufficient mechanistic understanding. In this issue of Cell, Ambrogio and colleagues connect the ability of K-Ras to dimerize to the ability of wild-type K-Ras to limit the oncogenic properties of the mutant.

Structural Insights into Non-canonical Ubiquitination Catalyzed by SidE.

Ubiquitination constitutes one of the most important signaling mechanisms in eukaryotes. Conventional ubiquitination is catalyzed by the universally conserved E1-E2-E3 three-enzyme cascade in an ATP-dependent manner. The newly identified SidE family effectors of the pathogen Legionella pneumophila ubiquitinate several human proteins by a different mechanism without engaging any of the conventional ubiquitination machinery. We now report the crystal structures of SidE alone and in complex with ubiquitin, NAD, and ADP-ribose, thereby capturing different conformations of SidE before and after ubiquitin and ligand binding. The structures of ubiquitin bound to both mART and PDE domains reveal several unique features of the two reaction steps catalyzed by SidE. Further, the structural and biochemical results demonstrate that SidE family members do not recognize specific structural folds of the substrate proteins. Our studies provide both structural explanations for the functional observations and new insights into the molecular mechanisms of this non-canonical ubiquitination machinery.

Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched.

Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.

Cryo-EM Reveals How Human Cytoplasmic Dynein Is Auto-inhibited and Activated.

Cytoplasmic dynein-1 binds dynactin and cargo adaptor proteins to form a transport machine capable of long-distance processive movement along microtubules. However, it is unclear why dynein-1 moves poorly on its own or how it is activated by dynactin. Here, we present a cryoelectron microscopy structure of the complete 1.4-megadalton human dynein-1 complex in an inhibited state known as the phi-particle. We reveal the 3D structure of the cargo binding dynein tail and show how self-dimerization of the motor domains locks them in a conformation with low microtubule affinity. Disrupting motor dimerization with structure-based mutagenesis drives dynein-1 into an open form with higher affinity for both microtubules and dynactin. We find the open form is also inhibited for movement and that dynactin relieves this by reorienting the motor domains to interact correctly with microtubules. Our model explains how dynactin binding to the dynein-1 tail directly stimulates its motor activity.

Decoupling the Functional Pleiotropy of Stem Cell Factor by Tuning c-Kit Signaling.

Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.

Molecular mechanism for target recognition, dimerization, and activation of Pyrococcus furiosus Argonaute.

The Argonaute nuclease from the thermophilic archaeon Pyrococcus furiosus (PfAgo) contributes to host defense and represents a promising biotechnology tool. Here, we report the structure of a PfAgo-guide DNA-target DNA ternary complex at the cleavage-compatible state. The ternary complex is predominantly dimerized, and the dimerization is solely mediated by PfAgo at PIWI-MID, PIWI-PIWI, and PAZ-N interfaces. Additionally, PfAgo accommodates a short 14-bp guide-target DNA duplex with a wedge-type N domain and specifically recognizes 5'-phosphorylated guide DNA. In contrast, the PfAgo-guide DNA binary complex is monomeric, and the engagement of target DNA with 14-bp complementarity induces sufficient dimerization and activation of PfAgo, accompanied by movement of PAZ and N domains. A closely related Argonaute from Thermococcus thioreducens adopts a similar dimerization configuration with an additional zinc finger formed at the dimerization interface. Dimerization of both Argonautes stabilizes the catalytic loops, highlighting the important role of Argonaute dimerization in the activation and target cleavage.

A protective Zika virus E-dimer-based subunit vaccine engineered to abrogate antibody-dependent enhancement of dengue infection.

Infections with dengue virus (DENV) and Zika virus (ZIKV) can induce cross-reactive antibody responses. Two immunodominant epitopes-one to precursor membrane protein and one to the fusion loop epitope on envelope (E) protein-are recognized by cross-reactive antibodies1-3 that are not only poorly neutralizing, but can also promote increased viral replication and disease severity via Fcγ receptor-mediated infection of myeloid cells-a process termed antibody-dependent enhancement (ADE)1,4,5. ADE is a significant concern for both ZIKV and DENV vaccines as the induction of poorly neutralizing cross-reactive antibodies may prime an individual for ADE on natural infection. In this report, we describe the design and production of covalently stabilized ZIKV E dimers, which lack precursor membrane protein and do not expose the immunodominant fusion loop epitope. Immunization of mice with ZIKV E dimers induces dimer-specific antibodies, which protect against ZIKV challenge during pregnancy. Importantly, the ZIKV E-dimer-induced response does not cross-react with DENV or induce ADE of DENV infection.

The transcription factor FoxP3 can fold into two dimerization states with divergent implications for regulatory T cell function and immune homeostasis.

FoxP3 is an essential transcription factor (TF) for immunologic homeostasis, but how it utilizes the common forkhead DNA-binding domain (DBD) to perform its unique function remains poorly understood. We here demonstrated that unlike other known forkhead TFs, FoxP3 formed a head-to-head dimer using a unique linker (Runx1-binding region [RBR]) preceding the forkhead domain. Head-to-head dimerization conferred distinct DNA-binding specificity and created a docking site for the cofactor Runx1. RBR was also important for proper folding of the forkhead domain, as truncation of RBR induced domain-swap dimerization of forkhead, which was previously considered the physiological form of FoxP3. Rather, swap-dimerization impaired FoxP3 function, as demonstrated with the disease-causing mutation R337Q, whereas a swap-suppressive mutation largely rescued R337Q-mediated functional impairment. Altogether, our findings suggest that FoxP3 can fold into two distinct dimerization states: head-to-head dimerization representing functional specialization of an ancient DBD and swap dimerization associated with impaired functions.

A Small Molecule RAS-Mimetic Disrupts RAS Association with Effector Proteins to Block Signaling.

Oncogenic activation of RAS genes via point mutations occurs in 20%-30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling.

Consensus on the RAS dimerization hypothesis: Strong evidence for lipid-mediated clustering but not for G-domain-mediated interactions.

One of the open questions in RAS biology is the existence of RAS dimers and their role in RAF dimerization and activation. The idea of RAS dimers arose from the discovery that RAF kinases function as obligate dimers, which generated the hypothesis that RAF dimer formation might be nucleated by G-domain-mediated RAS dimerization. Here, we review the evidence for RAS dimerization and describe a recent discussion among RAS researchers that led to a consensus that the clustering of two or more RAS proteins is not due to the stable association of G-domains but, instead, is a consequence of RAS C-terminal membrane anchors and the membrane phospholipids with which they interact.

A structural perspective on the regulation of the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a critical role in the pathogenesis of many cancers. The structure of intact forms of this receptor has yet to be determined, but intense investigations of fragments of the receptor have provided a detailed view of its activation mechanism, which we review here. Ligand binding converts the receptor to a dimeric form, in which contacts are restricted to the receptor itself, allowing heterodimerization of the four EGFR family members without direct ligand involvement. Activation of the receptor depends on the formation of an asymmetric dimer of kinase domains, in which one kinase domain allosterically activates the other. Coupling between the extracellular and intracellular domains may involve a switch between alternative crossings of the transmembrane helices, which form dimeric structures. We also discuss how receptor regulation is compromised by oncogenic mutations and the structural basis for negative cooperativity in ligand binding.

Advancing therapeutics using antibody-induced dimerization of receptor tyrosine phosphatases.

Receptor protein tyrosine phosphatases (RPTPs) are involved in a broad list of cellular, developmental, and physiological functions. Altering their expression leads to significant changes in protein phosphorylation linked to a growing list of human diseases, including cancers and neurological disorders. In this issue of Genes & Development, Qian and colleagues (pp. 743-759) present the identification of a monoclonal antibody targeting PTPRD extracellular domain-inducing dimerization and inhibition of the phosphatase activities, causing the proteolysis of dimeric PTPRD by a mechanism involving intracellular degradation pathways. Their study supports the potential of modulating PTPRD via its extracellular domains. This opens a new framework in the clinical manipulation of PTPRD and its closely related family members.

Manipulating PTPRD function with ectodomain antibodies.

Protein tyrosine phosphatases (PTPs) are critical regulators of signal transduction but have yet to be exploited fully for drug development. Receptor protein tyrosine phosphatase δ (RPTPδ/PTPRD) has been shown to elicit tumor-promoting functions, including elevating SRC activity and promoting metastasis in certain cell contexts. Dimerization has been implicated in the inhibition of receptor protein tyrosine phosphatases (RPTPs). We have generated antibodies targeting PTPRD ectodomains with the goal of manipulating their dimerization status ectopically, thereby regulating intracellular signaling. We have validated antibody binding to endogenous PTPRD in a metastatic breast cancer cell line, CAL51, and demonstrated that a monoclonal antibody, RD-43, inhibited phosphatase activity and induced the degradation of PTPRD. Similar effects were observed following chemically induced dimerization of its phosphatase domain. Mechanistically, RD-43 triggered the formation of PTPRD dimers in which the phosphatase activity was impaired. Subsequently, the mAb-PTPRD dimer complex was degraded through lysosomal and proteasomal pathways, independently of secretase cleavage. Consequently, treatment with RD-43 inhibited SRC signaling and suppressed PTPRD-dependent cell invasion. Together, these findings demonstrate that manipulating RPTP function via antibodies to the extracellular segments has therapeutic potential.

Functional Anatomy of the Human Microprocessor.

MicroRNA (miRNA) maturation is initiated by Microprocessor composed of RNase III DROSHA and its cofactor DGCR8, whose fidelity is critical for generation of functional miRNAs. To understand how Microprocessor recognizes pri-miRNAs, we here reconstitute human Microprocessor with purified recombinant proteins. We find that Microprocessor is an ∼364 kDa heterotrimeric complex of one DROSHA and two DGCR8 molecules. Together with a 23-amino acid peptide from DGCR8, DROSHA constitutes a minimal functional core. DROSHA serves as a "ruler" by measuring 11 bp from the basal ssRNA-dsRNA junction. DGCR8 interacts with the stem and apical elements through its dsRNA-binding domains and RNA-binding heme domain, respectively, allowing efficient and accurate processing. DROSHA and DGCR8, respectively, recognize the basal UG and apical UGU motifs, which ensure proper orientation of the complex. These findings clarify controversies over the action mechanism of DROSHA and allow us to build a general model for pri-miRNA processing.

Tuning cytokine receptor signaling by re-orienting dimer geometry with surrogate ligands.

Most cell-surface receptors for cytokines and growth factors signal as dimers, but it is unclear whether remodeling receptor dimer topology is a viable strategy to "tune" signaling output. We utilized diabodies (DA) as surrogate ligands in a prototypical dimeric receptor-ligand system, the cytokine Erythropoietin (EPO) and its receptor (EpoR), to dimerize EpoR ectodomains in non-native architectures. Diabody-induced signaling amplitudes varied from full to minimal agonism, and structures of these DA/EpoR complexes differed in EpoR dimer orientation and proximity. Diabodies also elicited biased or differential activation of signaling pathways and gene expression profiles compared to EPO. Non-signaling diabodies inhibited proliferation of erythroid precursors from patients with a myeloproliferative neoplasm due to a constitutively active JAK2V617F mutation. Thus, intracellular oncogenic mutations causing ligand-independent receptor activation can be counteracted by extracellular ligands that re-orient receptors into inactive dimer topologies. This approach has broad applications for tuning signaling output for many dimeric receptor systems.