ABSTRACT
Ligands of the Hedgehog (HH) pathway are paracrine signaling molecules that coordinate tissue development in metazoans. A remarkable feature of HH signaling is the repeated use of cholesterol in steps spanning ligand biogenesis, secretion, dispersal, and reception on target cells. A cholesterol molecule covalently attached to HH ligands is used as a molecular baton by transfer proteins to guide their secretion, spread, and reception. On target cells, a signaling circuit composed of a cholesterol transporter and sensor regulates transmission of HH signals across the plasma membrane to the cytoplasm. The repeated use of cholesterol in signaling supports the view that the HH pathway likely evolved by coopting ancient systems to regulate the abundance or organization of sterol-like lipids in membranes.
Subject(s)
Cholesterol , Hedgehog Proteins , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Ligands , Cholesterol/metabolism , Signal Transduction , Sterols/metabolismABSTRACT
During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a "trimer-of-trimers" super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.
Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , GPI-Linked Proteins/metabolism , Nerve Tissue Proteins/metabolism , Oncogene Proteins/metabolism , Animals , Cell Adhesion Molecules, Neuronal/chemistry , Cell Movement , DCC Receptor/deficiency , DCC Receptor/genetics , GPI-Linked Proteins/chemistry , Growth Cones/physiology , Humans , Lateral Ventricles/cytology , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/chemistry , Neurons/cytology , Neurons/metabolism , Oncogene Proteins/chemistry , Oncogene Proteins/genetics , Protein Binding , Protein Multimerization , Protein Structure, Quaternary , RNA Interference , RNA, Small Interfering/metabolism , Signal TransductionABSTRACT
The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.
Subject(s)
Acyltransferases/antagonists & inhibitors , Acyltransferases/metabolism , Enzyme Inhibitors/pharmacology , Hedgehog Proteins/metabolism , Membrane Proteins/metabolism , Acylation , Acyltransferases/genetics , Acyltransferases/ultrastructure , Allosteric Regulation , Animals , COS Cells , Catalytic Domain , Chlorocebus aethiops , Cryoelectron Microscopy , HEK293 Cells , Heme/metabolism , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Membrane Proteins/ultrastructure , Molecular Dynamics Simulation , Palmitoyl Coenzyme A/metabolism , Protein Conformation , Signal Transduction , Structure-Activity RelationshipABSTRACT
It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.
Subject(s)
Leukocyte Common Antigens/immunology , Receptors, Antigen, T-Cell/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology , Crystallography, X-Ray , HEK293 Cells , Humans , Jurkat Cells , Leukocyte Common Antigens/chemistry , Leukocyte Common Antigens/metabolism , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/immunology , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Microscopy, Electron , Microscopy, Fluorescence/methods , Models, Molecular , Protein Structure, Tertiary , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/metabolism , Time Factors , ZAP-70 Protein-Tyrosine Kinase/immunology , ZAP-70 Protein-Tyrosine Kinase/metabolismABSTRACT
A relatively small number of proteins have been suggested to act as morphogens-signalling molecules that spread within tissues to organize tissue repair and the specification of cell fate during development. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity1-3. How a hydrophobic lipoprotein can spread in the aqueous extracellular space is unknown. Several mechanisms, such as those involving lipoprotein particles, exosomes or a specific chaperone, have been proposed to overcome this so-called Wnt solubility problem4-6. Here we provide evidence against these models and show that the Wnt lipid is shielded by the core domain of a subclass of glypicans defined by the Dally-like protein (Dlp). Structural analysis shows that, in the presence of palmitoleoylated peptides, these glypicans change conformation to create a hydrophobic space. Thus, glypicans of the Dlp family protect the lipid of Wnt proteins from the aqueous environment and serve as a reservoir from which Wnt proteins can be handed over to signalling receptors.
Subject(s)
Glypicans/chemistry , Glypicans/metabolism , Lipids , Signal Transduction , Wnt Proteins/chemistry , Wnt Proteins/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Drosophila melanogaster , Fatty Acids, Monounsaturated/chemistry , Fatty Acids, Monounsaturated/metabolism , Female , Glypicans/classification , Humans , Hydrophobic and Hydrophilic Interactions , Lipids/chemistry , Male , Models, Molecular , Mutation , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding/genetics , Protein Domains , Protein Transport , Solubility , Wnt1 Protein/chemistry , Wnt1 Protein/metabolismABSTRACT
Primary cilia are required for Smoothened to transduce vertebrate Hedgehog signals, but how Smoothened accumulates in cilia and is activated is incompletely understood. Here, we identify cilia-associated oxysterols that promote Smoothened accumulation in cilia and activate the Hedgehog pathway. Our data reveal that cilia-associated oxysterols bind to two distinct Smoothened domains to modulate Smoothened accumulation in cilia and tune the intensity of Hedgehog pathway activation. We find that the oxysterol synthase HSD11ß2 participates in the production of Smoothened-activating oxysterols and promotes Hedgehog pathway activity. Inhibiting oxysterol biosynthesis impedes oncogenic Hedgehog pathway activation and attenuates the growth of Hedgehog pathway-associated medulloblastoma, suggesting that targeted inhibition of Smoothened-activating oxysterol production may be therapeutically useful for patients with Hedgehog-associated cancers.
Subject(s)
Cilia/drug effects , Cilia/metabolism , Oxysterols/pharmacology , Animals , Cell Line , HEK293 Cells , Hedgehog Proteins/metabolism , Humans , Mice , NIH 3T3 Cells , Signal Transduction/drug effectsABSTRACT
Repulsive guidance molecules (RGMs) are cell surface proteins that regulate the development and homeostasis of many tissues and organs, including the nervous, skeletal, and immune systems. They control fundamental biological processes, such as migration and differentiation by direct interaction with the Neogenin (NEO1) receptor and function as coreceptors for the bone morphogenetic protein (BMP)/growth differentiation factor (GDF) family. We determined crystal structures of all three human RGM family members in complex with GDF5, as well as the ternary NEO1-RGMB-GDF5 assembly. Surprisingly, we show that all three RGMs inhibit GDF5 signaling, which is in stark contrast to RGM-mediated enhancement of signaling observed for other BMPs, like BMP2. Despite their opposite effect on GDF5 signaling, RGMs occupy the BMP type 1 receptor binding site similar to the observed interactions in RGM-BMP2 complexes. In the NEO1-RGMB-GDF5 complex, RGMB physically bridges NEO1 and GDF5, suggesting cross-talk between the GDF5 and NEO1 signaling pathways. Our crystal structures, combined with structure-guided mutagenesis of RGMs and BMP ligands, binding studies, and cellular assays suggest that RGMs inhibit GDF5 signaling by competing with GDF5 type 1 receptors. While our crystal structure analysis and in vitro binding data initially pointed towards a simple competition mechanism between RGMs and type 1 receptors as a possible basis for RGM-mediated GDF5 inhibition, further experiments utilizing BMP2-mimicking GDF5 variants clearly indicate a more complex mechanism that explains how RGMs can act as a functionality-changing switch for two structurally and biochemically similar signaling molecules.
Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , GPI-Linked Proteins/metabolism , Growth Differentiation Factor 5/metabolism , Hemochromatosis Protein/metabolism , Nerve Tissue Proteins/metabolism , Bone Morphogenetic Protein 2/metabolism , Bone Morphogenetic Protein 2/ultrastructure , Cell Adhesion Molecules, Neuronal/ultrastructure , Crystallography, X-Ray , GPI-Linked Proteins/ultrastructure , Growth Differentiation Factor 5/ultrastructure , Hemochromatosis Protein/ultrastructure , Membrane Proteins/metabolism , Membrane Proteins/ultrastructure , Nerve Tissue Proteins/ultrastructure , Protein Domains , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Signal TransductionABSTRACT
Signaling pathways that mediate cell-cell communication are essential for collective cell behaviors in multicellular systems. The hedgehog (HH) pathway, first discovered and elucidated in Drosophila, is one of these iconic signaling systems that plays many roles during embryogenesis and in adults; abnormal HH signaling can lead to birth defects and cancer. We review recent structural and biochemical studies that have advanced our understanding of the vertebrate HH pathway, focusing on the mechanisms by which the HH signal is received by patched on target cells, transduced across the cell membrane by smoothened, and transmitted to the nucleus by GLI proteins to influence gene-expression programs.
Subject(s)
Hedgehog Proteins/metabolism , Vertebrates/metabolism , Animals , Hedgehog Proteins/genetics , Humans , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
The Hedgehog (Hh) signaling pathway coordinates cell-cell communication in development and regeneration. Defects in this pathway underlie diseases ranging from birth defects to cancer. Hh signals are transmitted across the plasma membrane by two proteins, Patched 1 (PTCH1) and Smoothened (SMO). PTCH1, a transporter-like tumor-suppressor protein, binds to Hh ligands, but SMO, a G-protein-coupled-receptor family oncoprotein, transmits the Hh signal across the membrane. Recent structural, biochemical and cell-biological studies have converged at the surprising model that a specific pool of plasma membrane cholesterol, termed accessible cholesterol, functions as a second messenger that conveys the signal between PTCH1 and SMO. Beyond solving a central puzzle in Hh signaling, these studies are revealing new principles in membrane biology: how proteins respond to and remodel cholesterol accessibility in membranes and how the cholesterol composition of organelle membranes is used to regulate protein function.
Subject(s)
Cell Membrane/metabolism , Cholesterol/metabolism , Cilia/metabolism , Hedgehog Proteins/metabolism , Patched-1 Receptor/metabolism , Smoothened Receptor/metabolism , Animals , Cell Membrane/chemistry , Cholesterol/chemistry , Cilia/chemistry , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Epithelial Cells/chemistry , Epithelial Cells/metabolism , Gene Expression Regulation , Hedgehog Proteins/chemistry , Hedgehog Proteins/genetics , Humans , Patched-1 Receptor/chemistry , Patched-1 Receptor/genetics , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Signal Transduction , Smoothened Receptor/chemistry , Smoothened Receptor/geneticsABSTRACT
Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzledclass G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked a top the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.
Subject(s)
Extracellular Space/metabolism , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Anilides/chemistry , Anilides/metabolism , Anilides/pharmacology , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Binding Sites/genetics , Cholesterol/metabolism , Cholesterol/pharmacology , Crystallography, X-Ray , Cysteine/chemistry , Cysteine/genetics , Cysteine/metabolism , Hedgehog Proteins/metabolism , Humans , Ligands , Models, Molecular , Protein Binding/genetics , Protein Stability/drug effects , Protein Structure, Tertiary/drug effects , Protein Structure, Tertiary/genetics , Pyridines/chemistry , Pyridines/metabolism , Pyridines/pharmacology , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/genetics , Signal Transduction/drug effects , Smoothened ReceptorABSTRACT
The most frequently mutated protein in human cancer is p53, a transcription factor (TF) that regulates myriad genes instrumental in diverse cellular outcomes including growth arrest and cell death. Cell context-dependent p53 modulation is critical for this life-or-death balance, yet remains incompletely understood. Here we identify sequence signatures enriched in genomic p53-binding sites modulated by the transcription cofactor iASPP. Moreover, our p53-iASPP crystal structure reveals that iASPP displaces the p53 L1 loop-which mediates sequence-specific interactions with the signature-corresponding base-without perturbing other DNA-recognizing modules of the p53 DNA-binding domain. A TF commonly uses multiple structural modules to recognize its cognate DNA, and thus this mechanism of a cofactor fine-tuning TF-DNA interactions through targeting a particular module is likely widespread. Previously, all tumor suppressors and oncoproteins that associate with the p53 DNA-binding domain-except the oncogenic E6 from human papillomaviruses (HPVs)-structurally cluster at the DNA-binding site of p53, complicating drug design. By contrast, iASPP inhibits p53 through a distinct surface overlapping the E6 footprint, opening prospects for p53-targeting precision medicine to improve cancer therapy.
Subject(s)
DNA/genetics , DNA/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Repressor Proteins/metabolism , Response Elements , Tumor Suppressor Protein p53/metabolism , Base Sequence , Binding Sites , Cell Line, Tumor , DNA/chemistry , Gene Expression Profiling , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Models, Molecular , Nucleotide Motifs , Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/metabolism , Protein Binding , Protein Conformation , Repressor Proteins/chemistry , Structure-Activity Relationship , Tumor Suppressor Protein p53/chemistryABSTRACT
Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids-a palmitoyl group at the N terminus and a cholesteroyl group at the C terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with reassessment of previous cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic hedgehog (Shh) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signaling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, Shh inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core.
Subject(s)
Hedgehog Proteins/metabolism , Patched-1 Receptor/metabolism , Animals , Cholesterol/chemistry , Gene Expression Regulation , HEK293 Cells , Hedgehog Proteins/chemistry , Hedgehog Proteins/genetics , Humans , Mice , Models, Molecular , NIH 3T3 Cells , Patched-1 Receptor/chemistry , Protein Binding , Protein Conformation , Single-Domain AntibodiesABSTRACT
The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small-molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT). Structure-activity relationship investigation identified single enantiomer IMP-1575, the most potent HHAT inhibitor reported to-date, and guided design of photocrosslinking probes that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed by kinetic analysis. Our results provide an optimal HHAT tool inhibitor IMP-1575 (Ki =38â nM) and a strategy for mapping small molecule interaction sites in MBOATs.
Subject(s)
Acetyltransferases/antagonists & inhibitors , Affinity Labels/chemistry , Small Molecule Libraries/chemistry , Acetyltransferases/metabolism , Binding Sites , Humans , Kinetics , Light , Palmitoyl Coenzyme A/antagonists & inhibitors , Palmitoyl Coenzyme A/metabolism , Pyridines/chemistry , Pyridines/metabolism , Small Molecule Libraries/metabolism , Structure-Activity RelationshipABSTRACT
The calcium-sensing receptor (CaSR) is a homodimeric G-protein-coupled receptor that signals via intracellular calcium (Ca2+i) mobilisation and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK) to regulate extracellular calcium (Ca2+e) homeostasis. The central importance of the CaSR in Ca2+e homeostasis has been demonstrated by the identification of loss- or gain-of-function CaSR mutations that lead to familial hypocalciuric hypercalcaemia (FHH) or autosomal dominant hypocalcaemia (ADH), respectively. However, the mechanisms determining whether the CaSR signals via Ca2+i or ERK have not been established, and we hypothesised that some CaSR residues, which are the site of both loss- and gain-of-function mutations, may act as molecular switches to direct signalling through these pathways. An analysis of CaSR mutations identified in >300 hypercalcaemic and hypocalcaemic probands revealed five 'disease-switch' residues (Gln27, Asn178, Ser657, Ser820 and Thr828) that are affected by FHH and ADH mutations. Functional expression studies using HEK293 cells showed disease-switch residue mutations to commonly display signalling bias. For example, two FHH-associated mutations (p.Asn178Asp and p.Ser820Ala) impaired Ca2+i signalling without altering ERK phosphorylation. In contrast, an ADH-associated p.Ser657Cys mutation uncoupled signalling by leading to increased Ca2+i mobilization while decreasing ERK phosphorylation. Structural analysis of these five CaSR disease-switch residues together with four reported disease-switch residues revealed these residues to be located at conformationally active regions of the CaSR such as the extracellular dimer interface and transmembrane domain. Thus, our findings indicate that disease-switch residues are located at sites critical for CaSR activation and play a role in mediating signalling bias.
Subject(s)
Gain of Function Mutation , Hypercalciuria/genetics , Hypocalcemia/genetics , Hypoparathyroidism/congenital , Loss of Function Mutation , Receptors, Calcium-Sensing/genetics , Signal Transduction , Amino Acid Sequence , Calcium Signaling , DNA Mutational Analysis , HEK293 Cells , Humans , Hypercalciuria/metabolism , Hypocalcemia/metabolism , Hypoparathyroidism/genetics , Hypoparathyroidism/metabolism , Protein Conformation , Receptors, Calcium-Sensing/metabolism , Sequence AlignmentABSTRACT
Hedgehog (Hh) proteins are secreted signaling molecules that mediate essential tissue-patterning events during embryonic development and function in tissue homeostasis and regeneration throughout life. Hh signaling is regulated by multiple mechanisms, including covalent lipid modification of the Hh protein and interactions with multiple protein and glycan partners. Unraveling the nature and effects of these interactions has proven challenging, but recent structural and biophysical studies of Hh proteins and active fragments of heparin, Ihog, Cdo, Boc, Hedgehog-interacting protein (Hhip), Patched (Ptc), and the monoclonal antibody 5E1 have added a new level of molecular detail to our understanding of how Hh signal response and distribution are regulated within tissues. We review these results and discuss their implications for understanding Hh signaling in normal and disease states.
Subject(s)
Hedgehog Proteins/chemistry , Hedgehog Proteins/metabolism , Animals , Binding Sites , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Models, Molecular , Patched Receptors , Protein Conformation , Protein Structure, Tertiary , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Signal TransductionABSTRACT
MHC class II molecules on the surface of antigen-presenting cells display a range of peptides for recognition by the T-cell receptors of CD4+ T helper cells. Therefore, MHC class II molecules are central to effective adaptive immune responses, but conversely, genetic and epidemiological data have implicated these molecules in the pathogenesis of autoimmune diseases. Indeed, the strength of the associations between particular MHC class II alleles and disease render them the main genetic risk factors for autoimmune disorders such as type 1 diabetes. Here, we discuss the insights that the crystal structures of MHC class II molecules provide into the molecular mechanisms by which sequence polymorphisms might contribute to disease susceptibility.
Subject(s)
Autoimmune Diseases/genetics , HLA-D Antigens/genetics , Autoimmune Diseases/immunology , Binding Sites/genetics , Binding Sites/immunology , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/metabolism , HLA-D Antigens/chemistry , HLA-D Antigens/metabolism , Humans , Models, Molecular , Protein Binding/genetics , Protein Binding/immunology , Protein ConformationABSTRACT
Cell-cell signalling of semaphorin ligands through interaction with plexin receptors is important for the homeostasis and morphogenesis of many tissues and is widely studied for its role in neural connectivity, cancer, cell migration and immune responses. SEMA4D and Sema6A exemplify two diverse vertebrate, membrane-spanning semaphorin classes (4 and 6) that are capable of direct signalling through members of the two largest plexin classes, B and A, respectively. In the absence of any structural information on the plexin ectodomain or its interaction with semaphorins the extracellular specificity and mechanism controlling plexin signalling has remained unresolved. Here we present crystal structures of cognate complexes of the semaphorin-binding regions of plexins B1 and A2 with semaphorin ectodomains (human PLXNB1(1-2)-SEMA4D(ecto) and murine PlxnA2(1-4)-Sema6A(ecto)), plus unliganded structures of PlxnA2(1-4) and Sema6A(ecto). These structures, together with biophysical and cellular assays of wild-type and mutant proteins, reveal that semaphorin dimers independently bind two plexin molecules and that signalling is critically dependent on the avidity of the resulting bivalent 2:2 complex (monomeric semaphorin binds plexin but fails to trigger signalling). In combination, our data favour a cell-cell signalling mechanism involving semaphorin-stabilized plexin dimerization, possibly followed by clustering, which is consistent with previous functional data. Furthermore, the shared generic architecture of the complexes, formed through conserved contacts of the amino-terminal seven-bladed ß-propeller (sema) domains of both semaphorin and plexin, suggests that a common mode of interaction triggers all semaphorin-plexin based signalling, while distinct insertions within or between blades of the sema domains determine binding specificity.
Subject(s)
Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Semaphorins/chemistry , Semaphorins/metabolism , Signal Transduction , Animals , Antigens, CD/chemistry , Antigens, CD/genetics , Antigens, CD/metabolism , Binding Sites , Cell Adhesion Molecules/genetics , Cell Communication , Crystallography, X-Ray , Humans , Ligands , Mice , Mice, Inbred C57BL , Models, Molecular , NIH 3T3 Cells , Nerve Tissue Proteins/genetics , Protein Binding , Protein Structure, Tertiary , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Semaphorins/genetics , Structure-Activity RelationshipABSTRACT
Hedgehog (Hh) morphogens play fundamental roles during embryogenesis and adulthood, in health and disease. Multiple cell surface receptors regulate the Hh signaling pathway. Among these, the glycosaminoglycan (GAG) chains of proteoglycans shape Hh gradients and signal transduction. We have determined crystal structures of Sonic Hh complexes with two GAGs, heparin and chondroitin sulfate. The interaction determinants, confirmed by site-directed mutagenesis and binding studies, reveal a previously not identified Hh site for GAG binding, common to all Hh proteins. The majority of Hh residues forming this GAG-binding site have been previously implicated in developmental diseases. Crystal packing analysis, combined with analytical ultracentrifugation of Sonic Hh-GAG complexes, suggests a potential mechanism for GAG-dependent Hh multimerization. Taken together, these results provide a direct mechanistic explanation of the observed correlation between disease and impaired Hh gradient formation. Moreover, GAG binding partially overlaps with the site of Hh interactions with an array of protein partners including Patched, hedgehog interacting protein, and the interference hedgehog protein family, suggesting a unique mechanism of Hh signaling modulation.
Subject(s)
Glycosaminoglycans/metabolism , Hedgehog Proteins/chemistry , Hedgehog Proteins/metabolism , Models, Molecular , Protein Conformation , Proteoglycans/metabolism , Signal Transduction/physiology , Chromatography, Affinity , Escherichia coli , Glycosaminoglycans/chemistry , Humans , Mutagenesis, Site-Directed , Polymerization , UltracentrifugationABSTRACT
Ten years ago nothing was known of the three-dimensional structure of members of the semaphorin family of cell guidance cues, nor of their major receptors, the plexins. The structural biology of this cell surface ligand-receptor system has now come of age. Detailed atomic level information is available on the architecture of semaphorin and plexin ectodomains and their recognition complexes. Similarly the structure of the plexin cytoplasmic region, and its interactions with members of the Rho family of small GTPases have been unveiled. These structural analyses, in combination with biochemical, biophysical and cellular studies, have progressed our understanding of this signalling system into the realm of molecular mechanism.
Subject(s)
Semaphorins/chemistry , Animals , Cytoplasm/chemistry , Cytoplasm/metabolism , Extracellular Space/chemistry , Extracellular Space/metabolism , Humans , Protein Binding , Semaphorins/metabolism , Signal TransductionABSTRACT
Plexins are cell surface receptors for the semaphorin family of cell guidance cues. The cytoplasmic region comprises a Ras GTPase-activating protein (GAP) domain and a RhoGTPase binding domain. Concomitant binding of extracellular semaphorin and intracellular RhoGTPase triggers GAP activity and signal transduction. The mechanism of this intricate regulation remains elusive. We present two crystal structures of the human Plexin-B1 cytoplasmic region in complex with a constitutively active RhoGTPase, Rac1. The structure of truncated Plexin-B1-Rac1 complex provides no mechanism for coupling RhoGTPase and Ras binding sites. On inclusion of the juxtamembrane helix, a trimeric structure of Plexin-B1-Rac1 complexes is stabilised by a second, novel, RhoGTPase binding site adjacent to the Ras site. Site-directed mutagenesis combined with cellular and biophysical assays demonstrate that this new binding site is essential for signalling. Our findings are consistent with a model in which extracellular and intracellular plexin clustering events combine into a single signalling output.