Structure of the thrombopoietin-MPL receptor complex is a blueprint for biasing hematopoiesis.

Thrombopoietin (THPO or TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Here, we report the 3.4 Å resolution cryoelectron microscopy structure of the extracellular TPO-TPO receptor (TpoR or MPL) signaling complex, revealing the basis for homodimeric MPL activation and providing a structural rationalization for genetic loss-of-function thrombocytopenia mutations. The structure guided the engineering of TPO variants (TPOmod) with a spectrum of signaling activities, from neutral antagonists to partial- and super-agonists. Partial agonist TPOmod decoupled JAK/STAT from ERK/AKT/CREB activation, driving a bias for megakaryopoiesis and platelet production without causing significant HSC expansion in mice and showing superior maintenance of human HSCs in vitro. These data demonstrate the functional uncoupling of the two primary roles of TPO, highlighting the potential utility of TPOmod in hematology research and clinical HSC transplantation.

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.

Functional Selectivity in Cytokine Signaling Revealed Through a Pathogenic EPO Mutation.

Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation (R150Q) in the cytokine erythropoietin (EPO). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild-type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.

Structure of the interleukin-5 receptor complex exemplifies the organizing principle of common beta cytokine signaling.

Cytokines regulate immune responses by binding to cell surface receptors, including the common subunit beta (ßc), which mediates signaling for GM-CSF, IL-3, and IL-5. Despite known roles in inflammation, the structural basis of IL-5 receptor activation remains unclear. We present the cryo-EM structure of the human IL-5 ternary receptor complex, revealing architectural principles for IL-5, GM-CSF, and IL-3. In mammalian cell culture, single-molecule imaging confirms hexameric IL-5 complex formation on cell surfaces. Engineered chimeric receptors show that IL-5 signaling, as well as IL-3 and GM-CSF, can occur through receptor heterodimerization, obviating the need for higher-order assemblies of ßc dimers. These findings provide insights into IL-5 and ßc receptor family signaling mechanisms, aiding in the development of therapies for diseases involving deranged ßc signaling.

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.

Organizing structural principles of the IL-17 ligand-receptor axis.

The IL-17 family of cytokines and receptors have central roles in host defence against infection and development of inflammatory diseases1. The compositions and structures of functional IL-17 family ligand-receptor signalling assemblies remain unclear. IL-17E (also known as IL-25) is a key regulator of type 2 immune responses and driver of inflammatory diseases, such as allergic asthma, and requires both IL-17 receptor A (IL-17RA) and IL-17RB to elicit functional responses2. Here we studied IL-25-IL-17RB binary and IL-25-IL-17RB-IL-17RA ternary complexes using a combination of cryo-electron microscopy, single-molecule imaging and cell-based signalling approaches. The IL-25-IL-17RB-IL-17RA ternary signalling assembly is a C2-symmetric complex in which the IL-25-IL-17RB homodimer is flanked by two 'wing-like' IL-17RA co-receptors through a 'tip-to-tip' geometry that is the key receptor-receptor interaction required for initiation of signal transduction. IL-25 interacts solely with IL-17RB to allosterically promote the formation of the IL-17RB-IL-17RA tip-to-tip interface. The resulting large separation between the receptors at the membrane-proximal level may reflect proximity constraints imposed by the intracellular domains for signalling. Cryo-electron microscopy structures of IL-17A-IL-17RA and IL-17A-IL-17RA-IL-17RC complexes reveal that this tip-to-tip architecture is a key organizing principle of the IL-17 receptor family. Furthermore, these studies reveal dual actions for IL-17RA sharing among IL-17 cytokine complexes, by either directly engaging IL-17 cytokines or alternatively functioning as a co-receptor.

Plasma membrane topography governs the 3D dynamic localization of IgM B cell antigen receptor clusters.

B lymphocytes recognize bacterial or viral antigens via different classes of the B cell antigen receptor (BCR). Protrusive structures termed microvilli cover lymphocyte surfaces, and are thought to perform sensory functions in screening antigen-bearing surfaces. Here, we have used lattice light-sheet microscopy in combination with tailored custom-built 4D image analysis to study the cell-surface topography of B cells of the Ramos Burkitt's Lymphoma line and the spatiotemporal organization of the IgM-BCR. Ramos B-cell surfaces were found to form dynamic networks of elevated ridges bridging individual microvilli. A fraction of membrane-localized IgM-BCR was found in clusters, which were mainly associated with the ridges and the microvilli. The dynamic ridge-network organization and the IgM-BCR cluster mobility were linked, and both were controlled by Arp2/3 complex activity. Our results suggest that dynamic topographical features of the cell surface govern the localization and transport of IgM-BCR clusters to facilitate antigen screening by B cells.

Structural linkage between ligand discrimination and receptor activation by type I interferons.

Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human type I IFN variants signal through the same cell-surface receptors, IFNAR1 and IFNAR2, yet they can evoke markedly different physiological effects. The crystal structures of two human type I IFN ternary signaling complexes containing IFNα2 and IFNω reveal recognition modes and heterotrimeric architectures that are unique among the cytokine receptor superfamily but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor points" interspersed among ligand-specific interactions that "tune" the relative IFN-binding affinities, in an apparent extracellular "ligand proofreading" mechanism that modulates biological activity. Functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively control signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns.

Structure of the IFNγ receptor complex guides design of biased agonists.

The cytokine interferon-γ (IFNγ) is a central coordinator of innate and adaptive immunity, but its highly pleiotropic actions have diminished its prospects for use as an immunotherapeutic agent. Here, we took a structure-based approach to decoupling IFNγ pleiotropy. We engineered an affinity-enhanced variant of the ligand-binding chain of the IFNγ receptor IFNγR1, which enabled us to determine the crystal structure of the complete hexameric (2:2:2) IFNγ-IFNγR1-IFNγR2 signalling complex at 3.25 Å resolution. The structure reveals the mechanism underlying deficits in IFNγ responsiveness in mycobacterial disease syndrome resulting from a T168N mutation in IFNγR2, which impairs assembly of the full signalling complex. The topology of the hexameric complex offers a blueprint for engineering IFNγ variants to tune IFNγ receptor signalling output. Unexpectedly, we found that several partial IFNγ agonists exhibited biased gene-expression profiles. These biased agonists retained the ability to induce upregulation of major histocompatibility complex class I antigen expression, but exhibited impaired induction of programmed death-ligand 1 expression in a wide range of human cancer cell lines, offering a route to decoupling immunostimulatory and immunosuppressive functions of IFNγ for therapeutic applications.

Dissociation of ß2m from MHC class I triggers formation of noncovalent transient heavy chain dimers.

At the plasma membrane of mammalian cells, major histocompatibility complex class I molecules (MHC-I) present antigenic peptides to cytotoxic T cells. Following the loss of the peptide and the light chain beta-2 microglobulin (ß2m, encoded by B2M), the resulting free heavy chains (FHCs) can associate into homotypic complexes in the plasma membrane. Here, we investigate the stoichiometry and dynamics of MHC-I FHCs assemblies by combining a micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single-molecule co-tracking. We identify non-covalent MHC-I FHC dimers, with dimerization mediated by the α3 domain, as the prevalent species at the plasma membrane, leading a moderate decrease in the diffusion coefficient. MHC-I FHC dimers show increased tendency to cluster into higher order oligomers as concluded from an increased immobile fraction with higher single-molecule colocalization. In vitro studies with isolated proteins in conjunction with molecular docking and dynamics simulations suggest that in the complexes, the α3 domain of one FHC binds to another FHC in a manner similar to that seen for ß2m.

Tuning MPL signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia progenitors.

Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable for both basic research and clinical utility. We developed a series of surrogate protein ligands for TPO-R, in the form of diabodies (DBs), that homodimerize TPO-R on the cell surface in geometries that are dictated by the DB receptor binding epitope, in effect "tuning" downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism, thus decoupling the dual functions of TPO/TPO-R. Using single-cell RNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in essential thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R.

Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis.

Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A "step-wise" preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB-promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II-TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.

Densely methylated DNA traps Methyl-CpG-binding domain protein 2 but permits free diffusion by Methyl-CpG-binding domain protein 3.

The methyl-CpG-binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands). Therefore, in this work, we investigate the binding and diffusion of MBD2 and MBD3 on more biologically relevant DNA templates that contain a large CpG island or limited CpG sites. Using a combination of single-molecule and biophysical analyses, we show that both MBD2 and MBD3 diffuse freely and rapidly across unmethylated CpG-rich DNA. In contrast, we found methylation of large CpG islands traps MBD2 leading to stable and apparently static binding on the CpG island while MBD3 continues to diffuse freely. In addition, we demonstrate both proteins bend DNA, which is augmented by methylation. Together, these studies support a model in which MBD2-NuRD strongly localizes to and compacts methylated CpG islands while MBD3-NuRD can freely mobilize nucleosomes independent of methylation status.

Surrogate Wnt agonists that phenocopy canonical Wnt and ß-catenin signalling.

Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing ß-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues. The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD-LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic ß-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine.

TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA.

The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2-TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein-DNA structures, and monitoring of DNA-DNA and DNA-RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA-dsDNA, dsDNA-ssDNA and dsDNA-ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.

Type I IFN is siloed in endosomes.

Type I IFN (IFN-I) is thought to be rapidly internalized and degraded following binding to its receptor and initiation of signaling. However, many studies report the persistent effects mediated by IFN-I for days or even weeks, both ex vivo and in vivo. These long-lasting effects are attributed to downstream signaling molecules or induced effectors having a long half-life, particularly in specific cell types. Here, we describe a mechanism explaining the long-term effects of IFN-I. Following receptor binding, IFN-I is siloed into endosomal compartments. These intracellular "IFN silos" persist for days and can be visualized by fluorescence and electron microscopy. However, they are largely dormant functionally, due to IFN-I-induced negative regulators. By contrast, in individuals lacking these negative regulators, such as ISG15 or USP18, this siloed IFN-I can continue to signal from within the endosome. This mechanism may underlie the long-term effects of IFN-I therapy and may contribute to the pathophysiology of type I interferonopathies.

Reversible Live-Cell Labeling with Retro-engineered HaloTags Enables Long-Term High- and Super-Resolution Imaging.

Self-labeling enzymes (SLE) such as the HaloTag have emerged as powerful tools in high and super-resolution fluorescence microscopy. Newly developed fluorogenic SLE substrates enable imaging in the presence of excess dye. To exploit this feature for reversible labeling, we engineered two variants of HaloTag7 with restored dehalogenase activity. Kinetic studies in vitro showed different turnover kinetics for reHaloTagS (≈0.006 s-1 ) and reHaloTagF (≈0.055 s-1 ). Imaging by confocal and stimulated emission depletion microscopy yielded 3-5-time enhanced photostability of reHaloTag labeling. Prominently, single molecule imaging with reHaloTags enabled controlled and stable labeling density over extended time periods. By combination with structured illumination, simultaneous visualization of single molecule diffusion and organellar dynamics was achieved. These applications highlight the potential of reHaloTag labeling for pushing the limits of advanced fluorescence microscopy techniques.

Flexible open conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi.

Vesicle formation at endomembranes requires the selective concentration of cargo by coat proteins. Conserved adapter protein complexes at the Golgi (AP-3), the endosome (AP-1), or the plasma membrane (AP-2) with their conserved core domain and flexible ear domains mediate this function. These complexes also rely on the small GTPase Arf1 and/or specific phosphoinositides for membrane binding. The structural details that influence these processes, however, are still poorly understood. Here we present cryo-EM structures of the full-length stable 300 kDa yeast AP-3 complex. The structures reveal that AP-3 adopts an open conformation in solution, comparable to the membrane-bound conformations of AP-1 or AP-2. This open conformation appears to be far more flexible than AP-1 or AP-2, resulting in compact, intermediate, and stretched subconformations. Mass spectrometrical analysis of the cross-linked AP-3 complex further indicates that the ear domains are flexibly attached to the surface of the complex. Using biochemical reconstitution assays, we also show that efficient AP-3 recruitment to the membrane depends primarily on cargo binding. Once bound to cargo, AP-3 clustered and immobilized cargo molecules, as revealed by single-molecule imaging on polymer-supported membranes. We conclude that its flexible open state may enable AP-3 to bind and collect cargo at the Golgi and could thus allow coordinated vesicle formation at the trans-Golgi upon Arf1 activation.

Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA.

TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation.

Biofunctional Nanodot Arrays in Living Cells Uncover Synergistic Co-Condensation of Wnt Signalodroplets.

Qualitative and quantitative analysis of transient signaling platforms in the plasma membrane has remained a key experimental challenge. Here, biofunctional nanodot arrays (bNDAs) are developed to spatially control dimerization and clustering of cell surface receptors at the nanoscale. High-contrast bNDAs with spot diameters of ≈300 nm are obtained by capillary nanostamping of bovine serum albumin bioconjugates, which are subsequently biofunctionalized by reaction with tandem anti-green fluorescence protein (GFP) clamp fusions. Spatially controlled assembly of active Wnt signalosomes is achieved at the nanoscale in the plasma membrane of live cells by capturing the co-receptor Lrp6 into bNDAs via an extracellular GFP tag. Strikingly, co-recruitment is observed of co-receptor Frizzled-8 as well as the cytosolic scaffold proteins Axin-1 and Disheveled-2 into Lrp6 nanodots in the absence of ligand. Density variation and the high dynamics of effector proteins uncover highly cooperative liquid-liquid phase separation (LLPS)-driven assembly of Wnt "signalodroplets" at the plasma membrane, pinpointing the synergistic effects of LLPS for Wnt signaling amplification. These insights highlight the potential of bNDAs for systematically interrogating nanoscale signaling platforms and condensation at the plasma membrane of live cells.