ABSTRACT
Human cytomegalovirus (HCMV) infects the majority of the human population and represents the leading viral cause of congenital birth defects. HCMV utilizes the glycoproteins gHgLgO (Trimer) to bind to platelet-derived growth factor receptor alpha (PDGFRα) and transforming growth factor beta receptor 3 (TGFßR3) to gain entry into multiple cell types. This complex is targeted by potent neutralizing antibodies and represents an important candidate for therapeutics against HCMV. Here, we determine three cryogenic electron microscopy (cryo-EM) structures of the trimer and the details of its interactions with four binding partners: the receptor proteins PDGFRα and TGFßR3 as well as two broadly neutralizing antibodies. Trimer binding to PDGFRα and TGFßR3 is mutually exclusive, suggesting that they function as independent entry receptors. In addition, Trimer-PDGFRα interaction has an inhibitory effect on PDGFRα signaling. Our results provide a framework for understanding HCMV receptor engagement, neutralization, and the development of anti-viral strategies against HCMV.
Subject(s)
Cytomegalovirus/chemistry , Membrane Glycoproteins/chemistry , Viral Envelope Proteins/chemistry , Virus Internalization , Cryoelectron Microscopy , Cytomegalovirus/physiology , Membrane Glycoproteins/metabolism , Models, Molecular , Proteoglycans/metabolism , Receptor, Platelet-Derived Growth Factor alpha/chemistry , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Receptors, Transforming Growth Factor beta/metabolism , Viral Envelope Proteins/metabolismABSTRACT
Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human Nav1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable â¼10 Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective Nav channel antagonists.
Subject(s)
NAV1.7 Voltage-Gated Sodium Channel/metabolism , NAV1.7 Voltage-Gated Sodium Channel/ultrastructure , Peptides/metabolism , Spider Venoms/metabolism , Amino Acid Sequence , Animals , Binding Sites , CHO Cells , Cricetulus , Cryoelectron Microscopy/methods , Crystallography, X-Ray/methods , HEK293 Cells , Humans , Ion Channel Gating , Peptides/toxicity , Protein Domains , Spider Venoms/toxicity , Spiders , Voltage-Gated Sodium Channel Blockers , Voltage-Gated Sodium Channels/metabolismABSTRACT
Characterizing cell surface receptors mediating viral infection is critical for understanding viral tropism and developing antiviral therapies. Nevertheless, due to challenges associated with detecting protein interactions on the cell surface, the host receptors of many human pathogens remain unknown. Here, we build a library consisting of most single transmembrane human receptors and implement a workflow for unbiased and high-sensitivity detection of receptor-ligand interactions. We apply this technology to elucidate the long-sought receptor of human cytomegalovirus (HCMV), the leading viral cause of congenital birth defects. We identify neuropilin-2 (Nrp2) as the receptor for HCMV-pentamer infection in epithelial/endothelial cells and uncover additional HCMV interactors. Using a combination of biochemistry, cell-based assays, and electron microscopy, we characterize the pentamer-Nrp2 interaction and determine the architecture of the pentamer-Nrp2 complex. This work represents an important approach to the study of host-pathogen interactions and provides a framework for understanding HCMV infection, neutralization, and the development of novel anti-HCMV therapies.
Subject(s)
Cytomegalovirus Infections/metabolism , Cytomegalovirus/physiology , Neuropilin-2/metabolism , Receptors, Virus/metabolism , Antibodies, Neutralizing/chemistry , Cell Membrane/metabolism , Endothelial Cells/metabolism , Epithelial Cells/metabolism , Epitope Mapping , Female , HEK293 Cells , Humans , Protein Conformation , Viral Envelope Proteins/metabolism , Virus InternalizationABSTRACT
Depolarizing sodium (Na+) leak currents carried by the NALCN channel regulate the resting membrane potential of many neurons to modulate respiration, circadian rhythm, locomotion and pain sensitivity1-8. NALCN requires FAM155A, UNC79 and UNC80 to function, but the role of these auxiliary subunits is not understood3,7,9-12. NALCN, UNC79 and UNC80 are essential in rodents2,9,13, and mutations in human NALCN and UNC80 cause severe developmental and neurological disease14,15. Here we determined the structure of the NALCN channelosome, an approximately 1-MDa complex, as fundamental aspects about the composition, assembly and gating of this channelosome remain obscure. UNC79 and UNC80 are massive HEAT-repeat proteins that form an intertwined anti-parallel superhelical assembly, which docks intracellularly onto the NALCN-FAM155A pore-forming subcomplex. Calmodulin copurifies bound to the carboxy-terminal domain of NALCN, identifying this region as a putative modulatory hub. Single-channel analyses uncovered a low open probability for the wild-type complex, highlighting the tightly closed S6 gate in the structure, and providing a basis to interpret the altered gating properties of disease-causing variants. Key constraints between the UNC79-UNC80 subcomplex and the NALCN DI-DII and DII-DIII linkers were identified, leading to a model of channelosome gating. Our results provide a structural blueprint to understand the physiology of the NALCN channelosome and a template for drug discovery to modulate the resting membrane potential.
Subject(s)
Ion Channels , Membrane Proteins , Amino Acid Motifs , Calmodulin , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Humans , Ion Channel Gating , Ion Channels/chemistry , Ion Channels/metabolism , Membrane Potentials , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Neurons/metabolism , Sodium/metabolismABSTRACT
Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response1-3. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein4-8, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1-/- macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1-/- mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.
Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , Cell Death , Cell Membrane/metabolism , Nerve Growth Factors/metabolism , Animals , Apoptosis , Cell Adhesion Molecules, Neuronal/chemistry , Cell Adhesion Molecules, Neuronal/genetics , Cell Death/genetics , Female , Humans , Macrophages , Male , Mice , Mutation , Necrosis , Nerve Growth Factors/chemistry , Nerve Growth Factors/genetics , Protein Multimerization , Pyroptosis/geneticsABSTRACT
Persistently depolarizing sodium (Na+) leak currents enhance electrical excitability1,2. The ion channel responsible for the major background Na+ conductance in neurons is the Na+ leak channel, non-selective (NALCN)3,4. NALCN-mediated currents regulate neuronal excitability linked to respiration, locomotion and circadian rhythm4-10. NALCN activity is under tight regulation11-14 and mutations in NALCN cause severe neurological disorders and early death15,16. NALCN is an orphan channel in humans, and fundamental aspects of channel assembly, gating, ion selectivity and pharmacology remain obscure. Here we investigate this essential leak channel and determined the structure of NALCN in complex with a distinct auxiliary subunit, family with sequence similarity 155 member A (FAM155A). FAM155A forms an extracellular dome that shields the ion-selectivity filter from neurotoxin attack. The pharmacology of NALCN is further delineated by a walled-off central cavity with occluded lateral pore fenestrations. Unusual voltage-sensor domains with asymmetric linkages to the pore suggest mechanisms by which NALCN activity is modulated. We found a tightly closed pore gate in NALCN where the majority of missense patient mutations cause gain-of-function phenotypes that cluster around the S6 gate and distinctive π-bulges. Our findings provide a framework to further study the physiology of NALCN and a foundation for discovery of treatments for NALCN channelopathies and other electrical disorders.
Subject(s)
Cryoelectron Microscopy , Ion Channels/chemistry , Ion Channels/ultrastructure , Membrane Proteins/chemistry , Membrane Proteins/ultrastructure , Gain of Function Mutation , HEK293 Cells , Humans , Ion Channels/genetics , Ion Channels/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Molecular , Mutation, Missense , Protein Domains , Protein Subunits/chemistry , Protein Subunits/metabolismABSTRACT
Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.
Subject(s)
Cell Membrane/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/chemistry , Escherichia coli/pathogenicity , Lipopolysaccharides/chemistry , Lipopolysaccharides/metabolism , Amidohydrolases/chemistry , Amidohydrolases/metabolism , Amino Acid Motifs , Bacterial Outer Membrane/chemistry , Bacterial Outer Membrane/metabolism , Binding Sites , Cell Membrane/metabolism , Enzyme Stability , Escherichia coli/cytology , Escherichia coli/drug effects , Genes, Essential , Hydrolases/chemistry , Hydrolases/metabolism , Lipid A/chemistry , Lipid A/metabolism , Lipopolysaccharides/biosynthesis , Microbial Sensitivity Tests , Microbial Viability/drug effects , Models, Molecular , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Periplasm/chemistry , Periplasm/metabolism , Protein Binding , VirulenceABSTRACT
The movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA. Recent structures of MsbA and related transporters have provided insights into the molecular basis of active lipid transport; however, structural information about their pharmacological modulation remains limited. Here we report the 2.9 Å resolution structure of MsbA in complex with G907, a selective small-molecule antagonist with bactericidal activity, revealing an unprecedented mechanism of ABC transporter inhibition. G907 traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains. This study establishes a framework for the selective modulation of ABC transporters and provides rational avenues for the design of new antibiotics and other therapeutics targeting this protein family.
Subject(s)
ATP-Binding Cassette Transporters/antagonists & inhibitors , ATP-Binding Cassette Transporters/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Quinolines/chemistry , Quinolines/pharmacology , ATP-Binding Cassette Transporters/metabolism , Allosteric Regulation/drug effects , Bacterial Proteins/metabolism , Binding Sites/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Escherichia coli/chemistry , Hydrocarbons/chemistry , Hydrocarbons/metabolism , Lipopolysaccharides/chemistry , Lipopolysaccharides/metabolism , Lipopolysaccharides/pharmacology , Models, Molecular , Protein Domains/drug effectsABSTRACT
Inflammasomes sense a number of pathogen and host damage signals to initiate a signaling cascade that triggers inflammatory cell death, termed pyroptosis. The inflammatory caspases (1/4/5/11) are the key effectors of this process through cleavage and activation of the pore-forming protein gasdermin D. Caspase-1 also activates proinflammatory interleukins, IL-1ß and IL-18, via proteolysis. However, compared to the well-studied apoptotic caspases, the identity of substrates and therefore biological functions of the inflammatory caspases remain limited. Here, we construct, validate, and apply an antibody toolset for direct detection of neo-C termini generated by inflammatory caspase proteolysis. By combining rabbit immune phage display with a set of degenerate and defined target peptides, we discovered two monoclonal antibodies that bind peptides with a similar degenerate recognition motif as the inflammatory caspases without recognizing the canonical apoptotic caspase recognition motif. Crystal structure analyses revealed the molecular basis of this strong yet paradoxical degenerate mode of peptide recognition. One antibody selectively immunoprecipitated cleaved forms of known and unknown inflammatory caspase substrates, allowing the identification of over 300 putative substrates of the caspase-4 noncanonical inflammasome, including caspase-7. This dataset will provide a path toward developing blood-based biomarkers of inflammasome activation. Overall, our study establishes tools to discover and detect inflammatory caspase substrates and functions, provides a workflow for designing antibody reagents to study cell signaling, and extends the growing evidence of biological cross talk between the apoptotic and inflammatory caspases.
Subject(s)
Amino Acid Motifs , Antibodies/chemistry , Antibodies/metabolism , Binding Sites , Caspases/metabolism , Inflammasomes/metabolism , Amino Acid Sequence , Caspases/chemistry , Models, Molecular , Peptides/chemistry , Peptides/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Proteolysis , Signal Transduction , Structure-Activity RelationshipABSTRACT
Antitumor immune responses depend on the infiltration of solid tumors by effector T cells, a process guided by chemokines. In particular, the chemokine CXCL10 has been shown to play a critical role in mediating recruitment of CXCR3 + cytolytic T and NK cells in tumors, though its use as a therapeutic agent has not been widely explored. One of the limitations is due to the rapid inactivation of CXCL10 by dipeptidyl peptidase 4 (DPP4), a broadly expressed enzyme that is active in plasma and other bodily fluids. In the present study, we describe a novel method to produce synthetic CXCL10 that is resistant to DPP4 N-terminal truncation. Using a Fmoc solid-phase peptide synthesis approach, synthetic murine WT CXCL10 was produced, showing similar biochemical and biological properties to the recombinant protein. This synthesis method supported production of natural (amino acid substitution, insertion or deletion) and non-natural (chemical modifications) variants of CXCL10. In association with a functional screening cascade that assessed DPP4-mediated cleavage, CXCR3 signaling potency and chemotactic activity, we successfully generated 20 murine CXCL10 variants. Among those, two non-natural variants with N-methylated Leu3 (MeLeu3) and a reduced amide bond between Pro2 and Leu3 (rLeu3), respectively, showed resistance to DPP4 truncation but decreased CXCR3 signaling and chemotactic activity. Interestingly, MeLeu3 and rLeu3 CXCL10 behaved as DPP4 inhibitors, preventing the truncation of WT CXCL10. This study highlights the potential of using Fmoc solid-phase chemistry in association with biochemical and biological characterization to rapidly identify CXCL10 variants with desired properties. These novel methods unlock the opportunity to develop DPP4 resistant CXCL10 variants, as well as other chemokine substrates, while maintaining chemotactic properties.
Subject(s)
Chemokine CXCL10/pharmacology , Dipeptidyl Peptidase 4/metabolism , Dipeptidyl-Peptidase IV Inhibitors/pharmacology , Chemokine CXCL10/chemical synthesis , Chemokine CXCL10/chemistry , Dipeptidyl-Peptidase IV Inhibitors/chemical synthesis , Dipeptidyl-Peptidase IV Inhibitors/chemistry , Dose-Response Relationship, Drug , Humans , Molecular Structure , Structure-Activity RelationshipABSTRACT
Prevailing dogma holds that cell-cell communication through Notch ligands and receptors determines binary cell fate decisions during progenitor cell divisions, with differentiated lineages remaining fixed. Mucociliary clearance in mammalian respiratory airways depends on secretory cells (club and goblet) and ciliated cells to produce and transport mucus. During development or repair, the closely related Jagged ligands (JAG1 and JAG2) induce Notch signalling to determine the fate of these lineages as they descend from a common proliferating progenitor. In contrast to such situations in which cell fate decisions are made in rapidly dividing populations, cells of the homeostatic adult airway epithelium are long-lived, and little is known about the role of active Notch signalling under such conditions. To disrupt Jagged signalling acutely in adult mammals, here we generate antibody antagonists that selectively target each Jagged paralogue, and determine a crystal structure that explains selectivity. We show that acute Jagged blockade induces a rapid and near-complete loss of club cells, with a concomitant gain in ciliated cells, under homeostatic conditions without increased cell death or division. Fate analyses demonstrate a direct conversion of club cells to ciliated cells without proliferation, meeting a conservative definition of direct transdifferentiation. Jagged inhibition also reversed goblet cell metaplasia in a preclinical asthma model, providing a therapeutic foundation. Our discovery that Jagged antagonism relieves a blockade of cell-to-cell conversion unveils unexpected plasticity, and establishes a model for Notch regulation of transdifferentiation.
Subject(s)
Antibodies/therapeutic use , Cell Transdifferentiation , Lung/cytology , Lung/metabolism , Receptors, Notch/metabolism , Animals , Antibodies/immunology , Antibodies/pharmacology , Asthma/drug therapy , Asthma/metabolism , Asthma/pathology , Calcium-Binding Proteins/antagonists & inhibitors , Calcium-Binding Proteins/immunology , Calcium-Binding Proteins/metabolism , Cell Death/drug effects , Cell Division/drug effects , Cell Lineage/drug effects , Cell Tracking , Cell Transdifferentiation/drug effects , Cilia/metabolism , Disease Models, Animal , Female , Goblet Cells/cytology , Goblet Cells/drug effects , Goblet Cells/pathology , Homeostasis/drug effects , Humans , Intercellular Signaling Peptides and Proteins/immunology , Intercellular Signaling Peptides and Proteins/metabolism , Jagged-1 Protein , Jagged-2 Protein , Ligands , Lung/drug effects , Male , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/immunology , Membrane Proteins/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Serrate-Jagged Proteins , Signal Transduction/drug effectsABSTRACT
Paired Immunoglobulin-like Type 2 Receptor Alpha (PILRA) is a cell surface inhibitory receptor that recognizes specific O-glycosylated proteins and is expressed on various innate immune cell types including microglia. We show here that a common missense variant (G78R, rs1859788) of PILRA is the likely causal allele for the confirmed Alzheimer's disease risk locus at 7q21 (rs1476679). The G78R variant alters the interaction of residues essential for sialic acid engagement, resulting in >50% reduced binding for several PILRA ligands including a novel ligand, complement component 4A, and herpes simplex virus 1 (HSV-1) glycoprotein B. PILRA is an entry receptor for HSV-1 via glycoprotein B, and macrophages derived from R78 homozygous donors showed significantly decreased levels of HSV-1 infection at several multiplicities of infection compared to homozygous G78 macrophages. We propose that PILRA G78R protects individuals from Alzheimer's disease risk via reduced inhibitory signaling in microglia and reduced microglial infection during HSV-1 recurrence.
Subject(s)
Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Genetic Variation , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Amino Acid Substitution , Animals , Genetic Loci , Humans , Ligands , Membrane Glycoproteins/chemistry , Mice , Models, Biological , Molecular Conformation , Protein Binding , Quantitative Trait Loci , Receptors, Immunologic/chemistry , Structure-Activity RelationshipABSTRACT
The folding and insertion of integral ß-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect ß-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the Escherichia coli ß-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its ß-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outer membrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying ß-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gram-negative bacterial growth by targeting extracellular epitopes.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antibodies, Bacterial/pharmacology , Antibodies, Monoclonal/pharmacology , Bacterial Outer Membrane Proteins/antagonists & inhibitors , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli/drug effects , Membrane Fluidity/drug effects , Bacterial Outer Membrane Proteins/immunology , Bacterial Outer Membrane Proteins/metabolism , Cell Membrane/drug effects , Cell Membrane/immunology , Cell Membrane/metabolism , Escherichia coli/immunology , Escherichia coli/metabolism , Escherichia coli Proteins/immunology , Escherichia coli Proteins/metabolism , Models, Molecular , Protein Conformation , Protein FoldingABSTRACT
Nitrate is a primary nutrient for plant growth, but its levels in soil can fluctuate by several orders of magnitude. Previous studies have identified Arabidopsis NRT1.1 as a dual-affinity nitrate transporter that can take up nitrate over a wide range of concentrations. The mode of action of NRT1.1 is controlled by phosphorylation of a key residue, Thr 101; however, how this post-translational modification switches the transporter between two affinity states remains unclear. Here we report the crystal structure of unphosphorylated NRT1.1, which reveals an unexpected homodimer in the inward-facing conformation. In this low-affinity state, the Thr 101 phosphorylation site is embedded in a pocket immediately adjacent to the dimer interface, linking the phosphorylation status of the transporter to its oligomeric state. Using a cell-based fluorescence resonance energy transfer assay, we show that functional NRT1.1 dimerizes in the cell membrane and that the phosphomimetic mutation of Thr 101 converts the protein into a monophasic high-affinity transporter by structurally decoupling the dimer. Together with analyses of the substrate transport tunnel, our results establish a phosphorylation-controlled dimerization switch that allows NRT1.1 to uptake nitrate with two distinct affinity modes.
Subject(s)
Anion Transport Proteins/chemistry , Arabidopsis/chemistry , Plant Proteins/chemistry , Protein Multimerization , Amino Acid Sequence , Anion Transport Proteins/genetics , Anion Transport Proteins/metabolism , Arabidopsis/genetics , Binding Sites , Biological Transport , Cell Membrane/chemistry , Cell Membrane/metabolism , Crystallography, X-Ray , Fluorescence Resonance Energy Transfer , Models, Biological , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Nitrate Transporters , Nitrates/chemistry , Nitrates/metabolism , Phosphorylation , Phosphothreonine/chemistry , Phosphothreonine/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Structure, Quaternary , Protons , Structure-Activity RelationshipABSTRACT
Voltage-gated calcium (CaV) channels catalyse rapid, highly selective influx of Ca(2+) into cells despite a 70-fold higher extracellular concentration of Na(+). How CaV channels solve this fundamental biophysical problem remains unclear. Here we report physiological and crystallographic analyses of a calcium selectivity filter constructed in the homotetrameric bacterial NaV channel NaVAb. Our results reveal interactions of hydrated Ca(2+) with two high-affinity Ca(2+)-binding sites followed by a third lower-affinity site that would coordinate Ca(2+) as it moves inward. At the selectivity filter entry, Site 1 is formed by four carboxyl side chains, which have a critical role in determining Ca(2+) selectivity. Four carboxyls plus four backbone carbonyls form Site 2, which is targeted by the blocking cations Cd(2+) and Mn(2+), with single occupancy. The lower-affinity Site 3 is formed by four backbone carbonyls alone, which mediate exit into the central cavity. This pore architecture suggests a conduction pathway involving transitions between two main states with one or two hydrated Ca(2+) ions bound in the selectivity filter and supports a 'knock-off' mechanism of ion permeation through a stepwise-binding process. The multi-ion selectivity filter of our CaVAb model establishes a structural framework for understanding the mechanisms of ion selectivity and conductance by vertebrate CaV channels.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Calcium Channels/chemistry , Calcium Channels/metabolism , Ion Channel Gating , Bacterial Proteins/genetics , Binding Sites , Biocatalysis , Calcium/metabolism , Calcium Channels/genetics , Cations, Divalent/metabolism , Crystallography, X-Ray , Electric Conductivity , Models, Biological , Models, Molecular , Structure-Activity Relationship , Substrate SpecificityABSTRACT
There is a critical need for new antibacterial strategies to counter the growing problem of antibiotic resistance. In Gram-negative bacteria, the outer membrane (OM) provides a protective barrier against antibiotics and other environmental insults. The outer leaflet of the outer membrane is primarily composed of lipopolysaccharide (LPS). Outer membrane biogenesis presents many potentially compelling drug targets as this pathway is absent in higher eukaryotes. Most proteins involved in LPS biosynthesis and transport are essential; however, few compounds have been identified that inhibit these proteins. The inner membrane ABC transporter MsbA carries out the first essential step in the trafficking of LPS to the outer membrane. We conducted a biochemical screen for inhibitors of MsbA and identified a series of quinoline compounds that kill Escherichia coli through inhibition of its ATPase and transport activity, with no loss of activity against clinical multidrug-resistant strains. Identification of these selective inhibitors indicates that MsbA is a viable target for new antibiotics, and the compounds we identified serve as useful tools to further probe the LPS transport pathway in Gram-negative bacteria.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Lipopolysaccharides/metabolism , Anti-Bacterial Agents/pharmacology , Biological Transport/drug effects , Biological Transport/physiology , Escherichia coli/drug effectsABSTRACT
In excitable cells, voltage-gated sodium (Na(V)) channels activate to initiate action potentials and then undergo fast and slow inactivation processes that terminate their ionic conductance. Inactivation is a hallmark of Na(V) channel function and is critical for control of membrane excitability, but the structural basis for this process has remained elusive. Here we report crystallographic snapshots of the wild-type Na(V)Ab channel from Arcobacter butzleri captured in two potentially inactivated states at 3.2 Å resolution. Compared to previous structures of Na(V)Ab channels with cysteine mutations in the pore-lining S6 helices (ref. 4), the S6 helices and the intracellular activation gate have undergone significant rearrangements: one pair of S6 helices has collapsed towards the central pore axis and the other S6 pair has moved outward to produce a striking dimer-of-dimers configuration. An increase in global structural asymmetry is observed throughout our wild-type Na(V)Ab models, reshaping the ion selectivity filter at the extracellular end of the pore, the central cavity and its residues that are analogous to the mammalian drug receptor site, and the lateral pore fenestrations. The voltage-sensing domains have also shifted around the perimeter of the pore module in wild-type Na(V)Ab, compared to the mutant channel, and local structural changes identify a conserved interaction network that connects distant molecular determinants involved in Na(V) channel gating and inactivation. These potential inactivated-state structures provide new insights into Na(V) channel gating and novel avenues to drug development and therapy for a range of debilitating Na(V) channelopathies.
Subject(s)
Arcobacter/chemistry , Ion Channel Gating , Sodium Channels/chemistry , Conserved Sequence , Crystallization , Crystallography, X-Ray , Models, Molecular , Protein Conformation , Sodium Channels/metabolism , Structure-Activity RelationshipABSTRACT
The voltage-gated sodium (Nav) channel Nav1.7 has been the focus of intense investigation in recent years. Human genetics studies of individuals with gain-of-function and loss-of-function mutations in the Nav1.7 channel have implicated Nav1.7 as playing a critical role in pain. Therefore, selective inhibition of Nav1.7 represents a potentially new analgesic strategy that is expected to be devoid of the significant liabilities associated with available treatment options. Although the identification and development of selective Nav channel modulators have historically been challenging, a number of recent publications has demonstrated progression of increasingly subtype-selective small molecules and peptides toward potential use in preclinical or clinical studies. In this respect, we focus on three binding sites that appear to offer the highest potential for the discovery and optimization of Nav1.7-selective inhibitors: the extracellular vestibule of the pore, the extracellular loops of voltage-sensor domain II (VSD2), and the extracellular loops of voltage-sensor domain IV (VSD4). Notably, these three receptor sites on Nav1.7 can all be defined as extracellular druggable sites, suggesting that non-small molecule formats are potential therapeutic options. In this chapter, we will review specific considerations and challenges underlying the identification and optimization of selective, potential therapeutics targeting Nav1.7 for chronic pain indications.