Higher-Order Clustering of the Transmembrane Anchor of DR5 Drives Signaling.

Receptor clustering on the cell membrane is critical in the signaling of many immunoreceptors, and this mechanism has previously been attributed to the extracellular and/or the intracellular interactions. Here, we report an unexpected finding that for death receptor 5 (DR5), a receptor in the tumor necrosis factor receptor superfamily, the transmembrane helix (TMH) alone in the receptor directly assembles a higher-order structure to drive signaling and that this structure is inhibited by the unliganded ectodomain. Nuclear magnetic resonance structure of the TMH in bicelles shows distinct trimerization and dimerization faces, allowing formation of dimer-trimer interaction networks. Single-TMH mutations that disrupt either trimerization or dimerization abolish ligand-induced receptor activation. Surprisingly, proteolytic removal of the DR5 ectodomain can fully activate downstream signaling in the absence of ligand. Our data suggest a receptor activation mechanism in which binding of ligand or antibodies to overcome the pre-ligand autoinhibition allows TMH clustering and thus signaling.

Molecular basis for interaction of let-7 microRNAs with Lin28.

MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate gene expression. Among these, members of the let-7 miRNA family control many cell-fate determination genes to influence pluripotency, differentiation, and transformation. Lin28 is a specific, posttranscriptional inhibitor of let-7 biogenesis. We report crystal structures of mouse Lin28 in complex with sequences from let-7d, let-7-f1, and let-7 g precursors. The two folded domains of Lin28 recognize two distinct regions of the RNA and are sufficient for inhibition of let-7 in vivo. We also show by NMR spectroscopy that the linker connecting the two folded domains is flexible, accommodating Lin28 binding to diverse let-7 family members. Protein-RNA complex formation imposes specific conformations on both components that could affect downstream recognition by other processing factors. Our data provide a molecular explanation for Lin28 specificity and a model for how it regulates let-7.

An amphipathic Bax core dimer forms part of the apoptotic pore wall in the mitochondrial␣membrane.

Bax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. This might involve their embedding in the cytosolic leaflet of the lipid bilayer, thus generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, Bax proteins might comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determined a high-resolution structure of the Bax core region, revealing a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but also creating polar interactions between charged residues and lipid heads. Structure-guided mutations demonstrate the importance of both types of protein-lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria.

Structural Basis and Functional Role of Intramembrane Trimerization of the Fas/CD95 Death Receptor.

Fas (CD95, Apo-1, or TNFRSF6) is a prototypical apoptosis-inducing death receptor in the tumor necrosis factor receptor (TNFR) superfamily. While the extracellular domains of TNFRs form trimeric complexes with their ligands and the intracellular domains engage in higher-order oligomerization, the role of the transmembrane (TM) domains is unknown. We determined the NMR structures of mouse and human Fas TM domains in bicelles that mimic lipid bilayers. Surprisingly, these domains use proline motifs to create optimal packing in homotrimer assembly distinct from classical trimeric coiled-coils in solution. Cancer-associated and structure-based mutations in Fas TM disrupt trimerization in vitro and reduce apoptosis induction in vivo, indicating the essential role of intramembrane trimerization in receptor activity. Our data suggest that the structures represent the signaling-active conformation of Fas TM, which appears to be different from the pre-ligand conformation. Analysis of other TNFR sequences suggests proline-containing sequences as common motifs for receptor TM trimerization.

Self-Assembled Antibody-Oligonucleotide Conjugates for Targeted Delivery of Complementary Antisense Oligonucleotides.

Antibody-oligonucleotide conjugate (AOC) affords preferential cell targeting and enhanced cellular uptake of antisense oligonucleotide (ASO).  Here, we have developed a modular AOC (MAOC) approach based on accurate self-assembly of separately prepared antibody and ASO modules. Homogeneous multimeric AOC with defined ASO-to-antibody ratio were generated by L-DNA scaffold mediated precise self-assembly of antibodies and ASOs. The MAOC approach has been implemented to deliver exon skipping ASOs via transferrin receptor (TfR1) mediated internalization. We discovered an anti-TfR1 sdAb that can greatly enhance nuclear delivery of ASOs. Cryo-EM structure of the sdAb-TfR1 complex showed a new epitope that does not overlap with the binding sites of endogenous TfR1 ligands. In vivo functional analyses of MAOCs with one ASO for single exon skipping and two ASOs for double exon skipping showed that both ASO concentration and exon skipping efficacy of MAOC in cardiac and skeletal muscles are dramatically higher than conventional ASOs in the transgenic Duchenne muscular dystrophy (DMD) mouse model. MAOC treatment was well tolerated in vivo and not associated with any toxicity-related morbidity or mortality. Collectively, our data suggest that the self-assembled MAOC is a viable option for broadening the therapeutic application of ASO via multi-specific targeting and delivery.

Regulation of T cell receptor activation by dynamic membrane binding of the CD3epsilon cytoplasmic tyrosine-based motif.

Many immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live-cell imaging studies showed a close interaction of the CD3epsilon cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3epsilon residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3epsilon ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation.

Self-Assembled L-DNA Linkers for Rapid Construction of Multi-Specific Antibody-Drug Conjugates Library.

One of the key challenges of improving clinical outcomes of antibody drug conjugates (ADCs) is overcoming cancer resistance to the antibody and/or drug components of ADCs, and hence the need for ADC platforms with high combinatory flexibility. Here, we introduce the use of self-assembled left-handed DNA (L-DNA) oligonucleotides to link combinatory single-domain antibodies and toxin payloads for tunable and adaptive delivery of ADCs. We demonstrate that the method allows convenient construction of a library of ADCs with multi-specific targeting, multi-specific payloads, and exact drug-antibody ratio. The newly constructed ADCs with L-DNA scaffold showed favorable properties of in vitro cell cytotoxicity and in vivo suppression and eradication of solid tumors. Collectively, our data suggest that the L-DNA based modular ADC (MADC) platform is a viable option for generating therapeutic ADCs and for potentially expanding ADC therapeutic window via multi-specificity.

Structure of the Streptococcus pyogenes NAD+ Glycohydrolase Translocation Domain and Its Essential Role in Toxin Binding to Oropharyngeal Keratinocytes.

The emergence and continued dominance of a Streptococcus pyogenes (group A Streptococcus, GAS) M1T1 clonal group is temporally correlated with acquisition of genomic sequences that confer high level expression of cotoxins streptolysin O (SLO) and NAD+-glycohydrolase (NADase). Experimental infection models have provided evidence that both toxins are important contributors to GAS virulence. SLO is a cholesterol-dependent pore-forming toxin capable of lysing virtually all types of mammalian cells. NADase, which is composed of an N-terminal translocation domain and C-terminal glycohydrolase domain, acts as an intracellular toxin that depletes host cell energy stores. NADase is dependent on SLO for internalization into epithelial cells, but its mechanism of interaction with the cell surface and details of its translocation mechanism remain unclear. In this study we found that NADase can bind oropharyngeal epithelial cells independently of SLO. This interaction is mediated by both domains of the toxin. We determined by NMR the structure of the translocation domain to be a ß-sandwich with a disordered N-terminal region. The folded region of the domain has structural homology to carbohydrate binding modules. We show that excess NADase inhibits SLO-mediated hemolysis and binding to epithelial cells in vitro, suggesting NADase and SLO have shared surface receptors. This effect is abrogated by disruption of a putative carbohydrate binding site on the NADase translocation domain. Our data are consistent with a model whereby interactions of the NADase glycohydrolase domain and translocation domain with SLO and the cell surface increase avidity of NADase binding and facilitate toxin-toxin and toxin-cell surface interactions. IMPORTANCE NADase and streptolysin O (SLO) are secreted toxins important for pathogenesis of group A Streptococcus, the agent of strep throat and severe invasive infections. The two toxins interact in solution and mutually enhance cytotoxic activity. We now find that NADase is capable of binding to the surface of human cells independently of SLO. Structural analysis of the previously uncharacterized translocation domain of NADase suggests that it contains a carbohydrate binding module. The NADase translocation domain and SLO appear to recognize similar glycan structures on the cell surface, which may be one mechanism through which NADase enhances SLO pore-forming activity during infection. Our findings provide new insight into the NADase toxin and its functional interactions with SLO during streptococcal infection.

The structural basis for intramembrane assembly of an activating immunoreceptor complex.

Many receptors that activate cells of the immune system are multisubunit membrane protein complexes in which ligand recognition and signaling functions are contributed by separate protein modules. Receptors and signaling subunits assemble through contacts among basic and acidic residues in their transmembrane domains to form the functional complexes. Here we report the nuclear magnetic resonance (NMR) structure of the membrane-embedded, heterotrimeric assembly formed by association of the DAP12 signaling module with the natural killer (NK) cell-activating receptor NKG2C. The main intramembrane contact site is formed by a complex electrostatic network involving five hydrophilic transmembrane residues. Functional mutagenesis demonstrated that similar polar intramembrane motifs are also important for assembly of the NK cell-activating NKG2D-DAP10 complex and the T cell antigen receptor (TCR)-invariant signaling protein CD3 complex. This structural motif therefore lies at the core of the molecular organization of many activating immunoreceptors.

HIV-1 fusion inhibitors targeting the membrane-proximal external region of Env spikes.

Combination antiretroviral therapy has transformed HIV-1 infection, once a fatal illness, into a manageable chronic condition. Drug resistance, severe side effects and treatment noncompliance bring challenges to combination antiretroviral therapy implementation in clinical settings and indicate the need for additional molecular targets. Here, we have identified several small-molecule fusion inhibitors, guided by a neutralizing antibody, against an extensively studied vaccine target-the membrane proximal external region (MPER) of the HIV-1 envelope spike. These compounds specifically inhibit the HIV-1 envelope-mediated membrane fusion by blocking CD4-induced conformational changes. An NMR structure of one compound complexed with a trimeric MPER construct reveals that the compound partially inserts into a hydrophobic pocket formed exclusively by the MPER residues, thereby stabilizing its prefusion conformation. These results suggest that the MPER is a potential therapeutic target for developing fusion inhibitors and that strategies employing an antibody-guided search for novel therapeutics may be applied to other human diseases.

Architecture of the mitochondrial calcium uniporter.

Mitochondria from many eukaryotic clades take up large amounts of calcium (Ca(2+)) via an inner membrane transporter called the uniporter. Transport by the uniporter is membrane potential dependent and sensitive to ruthenium red or its derivative Ru360 (ref. 1). Electrophysiological studies have shown that the uniporter is an ion channel with remarkably high conductance and selectivity. Ca(2+) entry into mitochondria is also known to activate the tricarboxylic acid cycle and seems to be crucial for matching the production of ATP in mitochondria with its cytosolic demand. Mitochondrial calcium uniporter (MCU) is the pore-forming and Ca(2+)-conducting subunit of the uniporter holocomplex, but its primary sequence does not resemble any calcium channel studied to date. Here we report the structure of the pore domain of MCU from Caenorhabditis elegans, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer in which the second transmembrane helix forms a hydrophilic pore across the membrane. The channel assembly represents a new solution of ion channel architecture, and is stabilized by a coiled-coil motif protruding into the mitochondrial matrix. The critical DXXE motif forms the pore entrance, which features two carboxylate rings; based on the ring dimensions and functional mutagenesis, these rings appear to form the selectivity filter. To our knowledge, this is one of the largest membrane protein structures characterized by NMR, and provides a structural blueprint for understanding the function of this channel.

A Trimeric Hydrophobic Zipper Mediates the Intramembrane Assembly of SARS-CoV-2 Spike.

The S protein of SARS-CoV-2 is a type I membrane protein that mediates membrane fusion and viral entry. A vast amount of structural information is available for the ectodomain of S, a primary target by the host immune system, but much less is known regarding its transmembrane domain (TMD) and its membrane-proximal regions. Here, we determined the NMR structure of the S protein TMD in bicelles that closely mimic a lipid bilayer. The TMD structure is a transmembrane α-helix (TMH) trimer that assembles spontaneously in a membrane. The trimer structure shows an extensive hydrophobic core along the 3-fold axis that resembles that of a trimeric leucine/isoleucine zipper, but with tetrad, not heptad, repeats. The trimeric core is strong in bicelles, resisting hydrogen-deuterium exchange for weeks. Although highly stable, structural guided mutagenesis identified single mutations that can completely dissociate the TMD trimer. Multiple studies have shown that the membrane anchors of viral fusion proteins can form highly specific oligomers, but the exact function of these oligomers remains unclear. Our findings should guide future experiments to address the above question for SARS coronaviruses.

NMR Model of the Entire Membrane-Interacting Region of the HIV-1 Fusion Protein and Its Perturbation of Membrane Morphology.

HIV-1 envelope glycoprotein (Env) is a transmembrane protein that mediates membrane fusion and viral entry. The membrane-interacting regions of the Env, including the membrane-proximal external region (MPER), the transmembrane domain (TMD), and the cytoplasmic tail (CT), not only are essential for fusion and Env incorporation but also can strongly influence the antigenicity of the Env. Previous studies have incrementally revealed the structures of the MPER, the TMD, and the KS-LLP2 regions of the CT. Here, we determined the NMR structure of the full-length CT using a protein fragment comprising the TMD and the CT in bicelles that mimic a lipid bilayer, and by integrating the new NMR data and those acquired previously on other gp41 fragments, we derived a model of the entire membrane-interacting region of the Env. The structure shows that the CT forms a large trimeric baseplate around the TMD trimer, and by residing in the headgroup region of the lipid bilayer, the baseplate causes severe exclusion of lipid in the cytoleaflet of the bilayer. All-atom molecular dynamics simulations showed that the overall structure of the MPER-TMD-CT can be stable in a viral membrane and that a concerted movement of the KS-LLP2 region compensates for the lipid exclusion in order to maintain both structure and membrane integrity. Our structural and simulation results provide a framework for future research to manipulate the membrane structure to modulate the antigenicity of the Env for vaccine development and for mutagenesis studies for investigating membrane fusion and Env interaction with the matrix proteins.

Structure of the membrane proximal external region of HIV-1 envelope glycoprotein.

The membrane-proximal external region (MPER) of the HIV-1 envelope glycoprotein (Env) bears epitopes of broadly neutralizing antibodies (bnAbs) from infected individuals; it is thus a potential vaccine target. We report an NMR structure of the MPER and its adjacent transmembrane domain in bicelles that mimic a lipid-bilayer membrane. The MPER lies largely outside the lipid bilayer. It folds into a threefold cluster, stabilized mainly by conserved hydrophobic residues and potentially by interaction with phospholipid headgroups. Antigenic analysis and comparison with published images from electron cryotomography of HIV-1 Env on the virion surface suggest that the structure may represent a prefusion conformation of the MPER, distinct from the fusion-intermediate state targeted by several well-studied bnAbs. Very slow bnAb binding indicates that infrequent fluctuations of the MPER structure give these antibodies occasional access to alternative conformations of MPER epitopes. Mutations in the MPER not only impede membrane fusion but also influence presentation of bnAb epitopes in other regions. These results suggest strategies for developing MPER-based vaccine candidates.

Inhibitor Development against p7 Channel in Hepatitis C Virus.

Hepatitis C Virus (HCV) is the key cause of chronic and severe liver diseases. The recent direct-acting antiviral agents have shown the clinical success on HCV-related diseases, but the rapid HCV mutations of the virus highlight the sustaining necessity to develop new drugs. p7, the viroporin protein from HCV, has been sought after as a potential anti-HCV drug target. Several classes of compounds, such as amantadine and rimantadine have been testified for p7 inhibition. However, the efficacies of these compounds are not high. Here, we screened some novel p7 inhibitors with amantadine scaffold for the inhibitor development. The dissociation constant (Kd) of 42 ARD-series compounds were determined by nuclear magnetic resonance (NMR) titrations. The efficacies of the two best inhibitors, ARD87 and ARD112, were further confirmed using viral production assay. The binding mode analysis and binding stability for the strongest inhibitor were deciphered by molecular dynamics (MD) simulation. These ARD-series compounds together with 49 previously published compounds were further analyzed by molecular docking. Key pharmacophores were identified among the structure-similar compounds. Our studies suggest that different functional groups are highly correlated with the efficacy for inhibiting p7 of HCV, in which hydrophobic interactions are the dominant forces for the inhibition potency. Our findings provide guiding principles for designing higher affinity inhibitors of p7 as potential anti-HCV drug candidates.

Ion and inhibitor binding of the double-ring ion selectivity filter of the mitochondrial calcium uniporter.

The calcium (Ca2+) uniporter of mitochondria is a holocomplex consisting of the Ca2+-conducting channel, known as mitochondrial calcium uniporter (MCU), and several accessory and regulatory components. A previous electrophysiology study found that the uniporter has high Ca2+ selectivity and conductance and this depends critically on the conserved amino acid sequence motif, DXXE (Asp-X-X-Glu) of MCU. A recent NMR structure of the MCU channel from Caenorhabditis elegans revealed that the DXXE forms two parallel carboxylate rings at the channel entrance that seem to serve as the ion selectivity filter, although direct ion interaction of this structural motif has not been addressed. Here, we use a paramagnetic probe, manganese (Mn2+), to investigate ion and inhibitor binding of this putative selectivity filter. Our paramagnetic NMR data show that mutants with a single carboxylate ring, NXXE (Asn-X-X-Glu) and DXXQ (Asp-X-X-Gln), each can bind Mn2+ specifically, whereas in the WT the two rings bind Mn2+ cooperatively, resulting in ∼1,000-fold higher apparent affinity. Ca2+ can specifically displace the bound Mn2+ at the DXXE site in the channel. Furthermore, titrating the sample with the known channel inhibitor ruthenium 360 (Ru360) can displace Mn2+ binding from the solvent-accessible Asp site but not the inner Glu site. The NMR titration data, together with structural analysis of the DXXE motif and molecular dynamics simulation, indicate that the double carboxylate rings at the apex of the MCU pore constitute the ion selectivity filter and that Ru360 directly blocks ion entry into the filter by binding to the outer carboxylate ring.

Critical Effect of the Detergent:Protein Ratio on the Formation of the Hepatitis C Virus p7 Channel.

The p7 protein encoded by the hepatitis C virus forms a cation-selective viroporin in the membrane. One of the most intriguing findings about the p7 viroporin is its unique hexameric structure in dodecylphosphocholine (DPC) micelles determined by nuclear magnetic resonance (NMR), but the hexameric structure was recently challenged by another NMR study of p7, also in DPC detergent, which claimed that the p7 in this detergent is monomeric. Here, we show that p7 oligomerization is highly sensitive to the detergent:protein ratio used in protein reconstitution and that the 40-fold difference in this ratio between the two studies was the cause of their different conclusions. In addition, we have performed extensive measurements of interchain paramagnetic relaxation enhancements (PREs) for p7 hexamers reconstituted in DPC micelles and in 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dihexanoyl-sn-glycero-3-phosphocholine bicelles. In both cases, interchain PREs are overall consistent with the hexameric structure determined in micelles. Our data validate the overall architecture of the p7 hexamer while highlighting the importance of the detergent:protein ratio in membrane protein sample preparation.

Identification of substrates of the small RNA methyltransferase Hen1 in mouse spermatogonial stem cells and analysis of its methyl-transfer domain.

Small noncoding RNAs (sncRNAs) regulate many genes in eukaryotic cells. Hua enhancer 1 (Hen1) is a 2'-O-methyltransferase that adds a methyl group to the 2'-OH of the 3'-terminal nucleotide of sncRNAs. The types and properties of sncRNAs may vary among different species, and the domain composition, structure, and function of Hen1 proteins differ accordingly. In mammals, Hen1 specifically methylates sncRNAs called P-element-induced wimpy testis-interacting RNAs (piRNAs). However, other types of sncRNAs that are methylated by Hen1 have not yet been reported, and the structures and the substrates of mammalian Hen1 remain unknown. Here, we report that mouse Hen1 (mHen1) performs 3'-end methylation of classical piRNAs, as well as those of most noncanonical piRNAs derived from rRNAs, small nuclear RNAs and tRNAs in murine spermatogonial stem cells. Moreover, we found that a distinct class of tRNA-derived sncRNAs are mHen1 substrates. We further determined the crystal structure of the putative methyltransferase domain of human Hen1 (HsHen1) in complex with its cofactor AdoMet at 2.0 Å resolution. We observed that HsHen1 has an active site similar to that of plant Hen1. We further found that the putative catalytic domain of HsHen1 alone exhibits no activity. However, an FXPP motif at its N terminus conferred full activity to this domain, and additional binding assays suggested that the FXPP motif is important for substrate binding. Our findings shed light on its methylation substrates in mouse spermatogonial stem cells and the substrate-recognition mechanism of mammalian Hen1.