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.

Structural basis of γ chain family receptor sharing at the membrane level.

Common γ chain (γc) cytokine receptors, including interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, are activated upon engagement with a common γc receptor (CD132) by concomitant binding of their ectodomains to an interleukin. In this work, we find that direct interactions between the transmembrane domains (TMDs) of both the γc and the interleukin receptors (ILRs) are also required for receptor activation. Moreover, the same γc TMD can specifically recognize multiple ILR TMDs of diverse sequences within the family. Heterodimer structures of γc TMD bound to IL-7 and IL-9 receptor TMDs-determined in a lipid bilayer-like environment by nuclear magnetic resonance spectroscopy-reveal a conserved knob-into-hole mechanism of recognition that mediates receptor sharing within the membrane. Thus, signaling in the γc receptor family requires specific heterotypic interactions of the TMDs.

Structural basis of γ -chain family receptor sharing at the membrane level.

The common γ-chain (γc) family of cytokine receptors, including interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, are activated upon engagement with the common γc receptor in ligand dependent manner. Sharing of γc by the IL receptors (ILRs) is thought to be achieved by concomitant binding of γc and ILR ectodomains to a cytokine. Here, we found that direct interactions between the transmembrane domain (TMD) of γc and those of the ILRs are also required for receptor activation, and remarkably, the same γc TMD can specifically recognize multiple ILR TMDs of diverse sequences. Heterodimer structures of γc TMD bound to the TMDs of IL-7R and IL-9R, determined in near lipid bilayer environment, reveal a conserved knob-into-hole mechanism of recognition that mediates receptor sharing within the membrane. Functional mutagenesis data indicate the requirement of the heterotypic interactions of TMDs in signaling, which could explain disease mutations within the receptor TMDs. One-Sentence Summary: The transmembrane anchors of interleukin receptors of the gamma-chain family are critical for receptor sharing and 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-guided and phage-assisted evolution of a therapeutic anti-EGFR antibody to reverse acquired resistance.

Acquired resistance to cetuximab in colorectal cancers is partially mediated by the acquisition of mutations located in the cetuximab epitope in the epidermal growth factor receptor (EGFR) ectodomain and hinders the clinical application of cetuximab. We develop a structure-guided and phage-assisted evolution approach for cetuximab evolution to reverse EGFRS492R- or EGFRG465R-driven resistance without altering the binding epitope or undermining antibody efficacy. Two evolved cetuximab variants, Ctx-VY and Ctx-Y104D, exhibit a restored binding ability with EGFRS492R, which harbors the most common resistance substitution, S492R. Ctx-W52D exhibits restored binding with EGFR harboring another common cetuximab resistance substitution, G465R (EGFRG465R). All the evolved cetuximab variants effectively inhibit EGFR activation and downstream signaling and induce the internalization and degradation of EGFRS492R and EGFRG465R as well as EGFRWT. The evolved cetuximab variants (Ctx-VY, Ctx-Y104D and Ctx-W52D) with one or two amino acid substitutions in the complementarity-determining region inherit the optimized physical and chemical properties of cetuximab to a great extent, thus ensuring their druggability. Our data collectively show that structure-guided and phage-assisted evolution is an efficient and general approach for reversing receptor mutation-mediated resistance to therapeutic antibody drugs.

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.

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.

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.

A trimeric hydrophobic zipper mediates the intramembrane assembly of SARS-CoV-2 spike.

The S protein of the 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 nuclear magnetic resonance (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 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, repeat. 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 anchor of viral fusion protein can form highly specific oligomers, but the exact function of these oligomers remain unclear. Our findings should guide future experiments to address the above question for SARS coronaviruses.

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.

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.

The Diversity and Similarity of Transmembrane Trimerization of TNF Receptors.

Receptors in the tumor necrosis factor receptor superfamily (TNFRSF) regulate proliferation of immune cells or induce programmed cell death, and many of them are candidates for antibody-based immunotherapy. Previous studies on several death receptors in the TNFRSF including Fas, p75NTR, and DR5 showed that the transmembrane helix (TMH) of these receptors can specifically oligomerize and their oligomeric states have direct consequences on receptor activation, suggesting a much more active role of TMH in receptor signaling than previously appreciated. Here, we report the structure of the TMH of TNFR1, another well studied member of the TNFRSF, in neutral bicelles that mimic a lipid bilayer. We find that TNFR1 TMH forms a defined trimeric complex in bicelles, and no evidences of higher-order clustering of trimers have been detected. Unexpectedly, a conserved proline, which is critical for Fas TMH trimerization, does not appear to play an important role in TNFR1 TMH trimerization, which is instead mediated by a glycine near the middle of the TMH. Further, TNFR1 TMH trimer shows a larger hydrophobic core than that of Fas or DR5, with four layers of hydrophobic interaction along the threefold axis. Comparison of the TNFR1 TMH structure with that of Fas and DR5 reveals reassuring similarities that have functional implications but also significant structural diversity that warrants systematic investigation of TMH oligomerization property for other members of the TNFRSF.

Structural basis of transmembrane coupling of the HIV-1 envelope glycoprotein.

The prefusion conformation of HIV-1 envelope protein (Env) is recognized by most broadly neutralizing antibodies (bnAbs). Studies showed that alterations of its membrane-related components, including the transmembrane domain (TMD) and cytoplasmic tail (CT), can reshape the antigenic structure of the Env ectodomain. Using nuclear magnetic resonance (NMR) spectroscopy, we determine the structure of an Env segment encompassing the TMD and a large portion of the CT in bicelles. The structure reveals that the CT folds into amphipathic helices that wrap around the C-terminal end of the TMD, thereby forming a support baseplate for the rest of Env. NMR dynamics measurements provide evidences of dynamic coupling across the TMD between the ectodomain and CT. Pseudovirus-based neutralization assays suggest that CT-TMD interaction preferentially affects antigenic structure near the apex of the Env trimer. These results explain why the CT can modulate the Env antigenic properties and may facilitate HIV-1 Env-based vaccine design.

Structural Characterization of the N-Terminal Domain of the Dictyostelium discoideum Mitochondrial Calcium Uniporter.

The mitochondrial calcium uniporter (MCU) plays a critical role in mitochondrial calcium uptake into the matrix. In metazoans, the uniporter is a tightly regulated multicomponent system, including the pore-forming subunit MCU and several regulators (MICU1, MICU2, and Essential MCU REgulator, EMRE). The calcium-conducting activity of metazoan MCU requires the single-transmembrane protein EMRE. Dictyostelium discoideum (Dd), however, developed a simplified uniporter for which the pore-forming MCU (DdMCU) alone is necessary and sufficient for calcium influx. Here, we report a crystal structure of the N-terminal domain (NTD) of DdMCU at 1.7 Å resolution. The DdMCU-NTD contains four helices and two strands arranged in a fold that is completely different from the known structures of other MCU-NTD homologues. Biochemical and biophysical analyses of DdMCU-NTD in solution indicated that the domain exists as high-order oligomers. Mutagenesis showed that the acidic residues Asp60, Glu72, and Glu74, which appeared to mediate the interface II, as observed in the crystal structure, participated in the self-assembly of DdMCU-NTD. Intriguingly, the oligomeric complex was disrupted in the presence of calcium. We propose that the calcium-triggered dissociation of NTD regulates the channel activity of DdMCU by a yet unknown mechanism.

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.

DNA-Mediated Assembly of Multispecific Antibodies for T Cell Engaging and Tumor Killing.

Targeting T-cells against cancer cells is a direct means of treating cancer, and has already shown great responses in clinical treatment of B-cell malignancies. A simple way to redirect T-cells to cancer cells is by using multispecific antibody (MsAb) that contains different arms for specifically "grabbing" the T-cells and cancer cells; as such, the T-cells are activated upon target engagement and the killing begins. Here, a nucleic acid mediated protein-protein assembly (NAPPA) approach is implemented to construct a MsAb for T-cell engaging and tumor killing. Anti -CD19 and -CD3 single-chain variable fragments (scFvs) are conjugated to different l-DNAs with sequences that form the Holliday junction, thus allowing spontaneous assembly of homogeneous protein-DNA oligomers containing two anti-CD19 and one anti-CD3 scFvs. The new MsAb shows strong efficacy in inducing Raji tumor cell cytotoxicity in the presence of T-cells with EC50 ≈ 0.2 × 10-9 m; it also suppresses tumor growth in a Raji xenograft mouse model. The data indicates that MsAbs assembled from protein-DNA conjugates are effective macromolecules for directing T-cells for tumor killing. The modular nature of the NAPPA platform allows rapid generation of complex MsAbs from simple antibody fragments, while offering a general solution for preparing antibodies with high-order specificity.

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.

Structure determination protocol for transmembrane domain oligomers.

The transmembrane (TM) anchors of cell surface proteins have been one of the 'blind spots' in structural biology because they are generally very hydrophobic, sometimes dynamic, and thus difficult targets for structural characterization. A plethora of examples show these membrane anchors are not merely anchors but can multimerize specifically to activate signaling receptors on the cell surface or to stabilize envelope proteins in viruses. Through a series of studies of the TM domains (TMDs) of immune receptors and viral membrane proteins, we have established a robust protocol for determining atomic-resolution structures of TM oligomers by NMR in bicelles that closely mimic a lipid bilayer. Our protocol overcomes hurdles typically encountered by structural biology techniques such as X-ray crystallography and cryo-electron microscopy (cryo-EM) when studying small TMDs. Here, we provide the details of the protocol, covering five major technical aspects: (i) a general method for producing isotopically labeled TM or membrane-proximal (MP) protein fragments that involves expression of the protein (which is fused to TrpLE) into inclusion bodies and releasing the target protein by cyanogen bromide (CNBr) cleavage; (ii) determination of the oligomeric state of TMDs in bicelles; (iii) detection of intermolecular contacts using nuclear Overhauser effect (NOE) experiments; (iv) structure determination; and (v) paramagnetic probe titration (PPT) to characterize the membrane partition of the TM oligomers. This protocol is broadly applicable for filling structural gaps of many type I/II membrane proteins. The procedures may take 3-6 months to complete, depending on the complexity and stability of the protein sample.

Unidirectional Presentation of Membrane Proteins in Nanoparticle-Supported Liposomes.

Presentation of membrane proteins to host immune systems has been a challenging problem owing to complexity arising from the poor in vivo stability of the membrane-mimetic media often used for solubilizing the membrane proteins. The use of functionalized, biocompatible nanoparticles as substrates is shown to guide the formation of proteoliposomes, which can present many copies of membrane proteins in a unidirectional manner. The approach was demonstrated to present the membrane-proximal region of the HIV-1 envelope glycoprotein. These nanoparticle-supported liposomes are broadly applicable as membrane antigen vehicles for inducing host immune responses.