EGFR Ligands Differentially Stabilize Receptor Dimers to Specify Signaling Kinetics.

Epidermal growth factor receptor (EGFR) regulates many crucial cellular programs, with seven different activating ligands shaping cell signaling in distinct ways. Using crystallography and other approaches, we show how the EGFR ligands epiregulin (EREG) and epigen (EPGN) stabilize different dimeric conformations of the EGFR extracellular region. As a consequence, EREG or EPGN induce less stable EGFR dimers than EGF-making them partial agonists of EGFR dimerization. Unexpectedly, this weakened dimerization elicits more sustained EGFR signaling than seen with EGF, provoking responses in breast cancer cells associated with differentiation rather than proliferation. Our results reveal how responses to different EGFR ligands are defined by receptor dimerization strength and signaling dynamics. These findings have broad implications for understanding receptor tyrosine kinase (RTK) signaling specificity. Our results also suggest parallels between partial and/or biased agonism in RTKs and G-protein-coupled receptors, as well as new therapeutic opportunities for correcting RTK signaling output.

Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation.

Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 Å resolution cryoelectron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses.

Prefusion stabilization of the Hendra and Langya virus F proteins.

Nipah virus (NiV) and Hendra virus (HeV) are pathogenic paramyxoviruses that cause mild-to-severe disease in humans. As members of the Henipavirus genus, NiV and HeV use an attachment (G) glycoprotein and a class I fusion (F) glycoprotein to invade host cells. The F protein rearranges from a metastable prefusion form to an extended postfusion form to facilitate host cell entry. Prefusion NiV F elicits higher neutralizing antibody titers than postfusion NiV F, indicating that stabilization of prefusion F may aid vaccine development. A combination of amino acid substitutions (L104C/I114C, L172F, and S191P) is known to stabilize NiV F in its prefusion conformation, although the extent to which substitutions transfer to other henipavirus F proteins is not known. Here, we perform biophysical and structural studies to investigate the mechanism of prefusion stabilization in F proteins from three henipaviruses: NiV, HeV, and Langya virus (LayV). Three known stabilizing substitutions from NiV F transfer to HeV F and exert similar structural and functional effects. One engineered disulfide bond, located near the fusion peptide, is sufficient to stabilize the prefusion conformations of both HeV F and LayV F. Although LayV F shares low overall sequence identity with NiV F and HeV F, the region around the fusion peptide exhibits high sequence conservation across all henipaviruses. Our findings indicate that substitutions targeting this site of conformational change might be applicable to prefusion stabilization of other henipavirus F proteins and support the use of NiV as a prototypical pathogen for henipavirus vaccine antigen design.IMPORTANCEPathogenic henipaviruses such as Nipah virus (NiV) and Hendra virus (HeV) cause respiratory symptoms, with severe cases resulting in encephalitis, seizures, and coma. The work described here shows that the NiV and HeV fusion (F) proteins share common structural features with the F protein from an emerging henipavirus, Langya virus (LayV). Sequence alignment alone was sufficient to predict which known prefusion-stabilizing amino acid substitutions from NiV F would stabilize the prefusion conformations of HeV F and LayV F. This work also reveals an unexpected oligomeric interface shared by prefusion HeV F and NiV F. Together, these advances lay a foundation for future antigen design targeting henipavirus F proteins. In this way, Nipah virus can serve as a prototypical pathogen for the development of protective vaccines and monoclonal antibodies to prepare for potential henipavirus outbreaks.

Distinctive features of the respiratory syncytial virus priming loop compared to other non-segmented negative strand RNA viruses.

De novo initiation by viral RNA-dependent RNA polymerases often requires a polymerase priming residue, located within a priming loop, to stabilize the initiating NTPs. Polymerase structures from three different non-segmented negative strand RNA virus (nsNSV) families revealed putative priming loops in different conformations, and an aromatic priming residue has been identified in the rhabdovirus polymerase. In a previous study of the respiratory syncytial virus (RSV) polymerase, we found that Tyr1276, the L protein aromatic amino acid residue that most closely aligns with the rhabdovirus priming residue, is not required for RNA synthesis but two nearby residues, Pro1261 and Trp1262, were required. In this study, we examined the roles of Pro1261 and Trp1262 in RNA synthesis initiation. Biochemical studies showed that substitution of Pro1261 inhibited RNA synthesis initiation without inhibiting back-priming, indicating a defect in initiation. Biochemical and minigenome experiments showed that the initiation defect incurred by a P1261A substitution could be rescued by factors that would be expected to increase the stability of the initiation complex, specifically increased NTP concentration, manganese, and a more efficient promoter sequence. These findings indicate that Pro1261 of the RSV L protein plays a role in initiation, most likely in stabilizing the initiation complex. However, we found that substitution of the corresponding proline residue in a filovirus polymerase had no effect on RNA synthesis initiation or elongation. These results indicate that despite similarities between the nsNSV polymerases, there are differences in the features required for RNA synthesis initiation.

Activity of EGFR transmembrane region variants indicates specific transmembrane dimers are not required for EGFR activity.

The Epidermal Growth Factor Receptor (EGFR) is a Receptor Tyrosine Kinase that mediates cell proliferation and differentiation events during development and maintenance of complex organisms. Formation of specific, ligand-dependent EGFR dimers is a key step in stimulating EGFR signaling, and crystal structures of active, dimeric forms of isolated EGFR extracellular regions and kinase domains have revealed much about how dimer interactions regulate EGFR activity. The nature and role of the transmembrane region in regulating EGFR activity remains less clear, however. Proposed roles for the transmembrane region range from nonspecific but energetically favorable interactions to specific transmembrane dimer conformations being associated with active, inactive, or activity-modulated states of EGFR. To investigate the role of specific transmembrane dimers in modulating EGFR activity we generated thirteen EGFR variants with altered transmembrane sequences designed to favor or disfavor specific types of transmembrane region interactions. We show using FRET microscopy that EGFR transmembrane regions have an intrinsic propensity to associate in mammalian cell membranes that is counteracted by the extracellular region. We show using cell-based assays that each of the EGFR transmembrane variants except the Neu variant, which results in constitutive receptor phosphorylation, is able to autophosphorylate and stimulate phosphorylation of downstream effectors Erk and Akt. Our results indicate that many transmembrane sequences, including polyleucine, are compatible with EGFR activity and provide no evidence for specific transmembrane dimers regulating EGFR function.

EGFR forms ligand-independent oligomers that are distinct from the active state.

The human epidermal growth factor receptor (EGFR/ERBB1) is a receptor tyrosine kinase (RTK) that forms activated oligomers in response to ligand. Much evidence indicates that EGFR/ERBB1 also forms oligomers in the absence of ligand, but the structure and physiological role of these ligand-independent oligomers remain unclear. To examine these features, we use fluorescence microscopy to measure the oligomer stability and FRET efficiency for homo- and hetero-oligomers of fluorescent protein-labeled forms of EGFR and its paralog, human epidermal growth factor receptor 2 (HER2/ERBB2) in vesicles derived from mammalian cell membranes. We observe that both receptors form ligand-independent oligomers at physiological plasma membrane concentrations. Mutations introduced in the kinase region at the active state asymmetric kinase dimer interface do not affect the stability of ligand-independent EGFR oligomers. These results indicate that ligand-independent EGFR oligomers form using interactions that are distinct from the EGFR active state.

A single ligand is sufficient to activate EGFR dimers.

Crystal structures of human epidermal growth factor receptor (EGFR) with bound ligand revealed symmetric, doubly ligated receptor dimers thought to represent physiologically active states. Such complexes fail to rationalize negative cooperativity of epidermal growth factor (EGF) binding to EGFR and the behavior of the ligandless EGFR homolog ErbB2/HER2, however. We report cell-based assays that provide evidence for active, singly ligated dimers of human EGFR and its homolog, ErbB4/HER4. We also report crystal structures of the ErbB4/HER4 extracellular region complexed with its ligand Neuregulin-1ß that resolve two types of ErbB dimer when compared to EGFR:Ligand complexes. One type resembles the recently reported asymmetric dimer of Drosophila EGFR with a single high-affinity ligand bound and provides a model for singly ligated human ErbB dimers. These results unify models of vertebrate and invertebrate EGFR/ErbB signaling, imply that the tethered conformation of unliganded ErbBs evolved to prevent crosstalk among ErbBs, and establish a molecular basis for both negative cooperativity of ligand binding to vertebrate ErbBs and the absence of active ErbB2/HER2 homodimers in normal conditions.

Hybrid immunity to SARS-CoV-2 arises from serological recall of IgG antibodies distinctly imprinted by infection or vaccination.

We used plasma IgG proteomics to study the molecular composition and temporal durability of polyclonal IgG antibodies triggered by ancestral SARS-CoV-2 infection, vaccination, or their combination ("hybrid immunity"). Infection, whether primary or post-vaccination, mainly triggered an anti-spike antibody response to the S2 domain, while vaccination predominantly induced anti-RBD antibodies. Immunological imprinting persisted after a secondary (hybrid) exposure, with >60% of the ensuing serological response originating from the initial antibodies generated during the first exposure. We highlight one instance where hybrid immunity arising from breakthrough infection resulted in a marked increase in the breadth and affinity of a highly abundant vaccination-elicited plasma IgG antibody, SC27. With an intrinsic binding affinity surpassing a theoretical maximum (K D < 5 pM), SC27 demonstrated potent neutralization of various SARS-CoV-2 variants and SARS-like zoonotic viruses (IC 50 ∼0.1-1.75 nM) and provided robust protection in vivo . Cryo-EM structural analysis unveiled that SC27 binds to the RBD class 1/4 epitope, with both VH and VL significantly contributing to the binding interface. These findings suggest that exceptionally broad and potent antibodies can be prevalent in plasma and can largely dictate the nature of serological neutralization. HIGHLIGHTS: ▪ Infection and vaccination elicit unique IgG antibody profiles at the molecular level▪ Immunological imprinting varies between infection (S2/NTD) and vaccination (RBD)▪ Hybrid immunity maintains the imprint of first infection or first vaccination▪ Hybrid immune IgG plasma mAbs have superior neutralization potency and breadth.

Hybrid immunity to SARS-CoV-2 arises from serological recall of IgG antibodies distinctly imprinted by infection or vaccination.

We describe the molecular-level composition of polyclonal immunoglobulin G (IgG) anti-spike antibodies from ancestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, vaccination, or their combination ("hybrid immunity") at monoclonal resolution. Infection primarily triggers S2/N-terminal domain (NTD)-reactive antibodies, whereas vaccination mainly induces anti-receptor-binding domain (RBD) antibodies. This imprint persists after secondary exposures wherein >60% of ensuing hybrid immunity derives from the original IgG pool. Monoclonal constituents of the original IgG pool can increase breadth, affinity, and prevalence upon secondary exposures, as exemplified by the plasma antibody SC27. Following a breakthrough infection, vaccine-induced SC27 gained neutralization breadth and potency against SARS-CoV-2 variants and zoonotic viruses (half-maximal inhibitory concentration [IC50] ∼0.1-1.75 nM) and increased its binding affinity to the protective RBD class 1/4 epitope (dissociation constant [KD] < 5 pM). According to polyclonal escape analysis, SC27-like binding patterns are common in SARS-CoV-2 hybrid immunity. Our findings provide a detailed molecular definition of immunological imprinting and show that vaccination can produce class 1/4 (SC27-like) IgG antibodies circulating in the blood.

Structural basis for antibody recognition of vulnerable epitopes on Nipah virus F protein.

Nipah virus (NiV) is a pathogenic paramyxovirus that causes fatal encephalitis in humans. Two envelope glycoproteins, the attachment protein (G/RBP) and fusion protein (F), facilitate entry into host cells. Due to its vital role, NiV F presents an attractive target for developing vaccines and therapeutics. Several neutralization-sensitive epitopes on the NiV F apex have been described, however the antigenicity of most of the F protein's surface remains uncharacterized. Here, we immunize mice with prefusion-stabilized NiV F and isolate ten monoclonal antibodies that neutralize pseudotyped virus. Cryo-electron microscopy reveals eight neutralization-sensitive epitopes on NiV F, four of which have not previously been described. Novel sites span the lateral and basal faces of NiV F, expanding the known library of vulnerable epitopes. Seven of ten antibodies bind the Hendra virus (HeV) F protein. Multiple sequence alignment suggests that some of these newly identified neutralizing antibodies may also bind F proteins across the Henipavirus genus. This work identifies new epitopes as targets for therapeutics, provides a molecular basis for NiV neutralization, and lays a foundation for development of new cross-reactive antibodies targeting Henipavirus F proteins.

Principles and practical applications of structure-based vaccine design.

Viral proteins fold into a variety of structures as they perform their functions. Structure-based vaccine design aims to exploit knowledge of an antigen's architecture to stabilize it in a vulnerable conformation. We summarize the general principles of structure-based vaccine design, with a focus on the major types of sequence modifications: proline, disulfide, cavity-filling, electrostatic and hydrogen-bond substitution, as well as domain deletion. We then review recent applications of these principles to vaccine-design efforts across five viral families: Coronaviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, and Filoviridae. Outstanding challenges include continued application of proven design principles to pathogens of interest, as well as development of new strategies for those pathogens that resist traditional techniques.

IgG-like bispecific antibodies with potent and synergistic neutralization against circulating SARS-CoV-2 variants of concern.

Monoclonal antibodies are a promising approach to treat COVID-19, however the emergence of SARS-CoV-2 variants has challenged the efficacy and future of these therapies. Antibody cocktails are being employed to mitigate these challenges, but neutralization escape remains a major challenge and alternative strategies are needed. Here we present two anti-SARS-CoV-2 spike binding antibodies, one Class 1 and one Class 4, selected from our non-immune human single-chain variable fragment (scFv) phage library, that are engineered into four, fully-human IgG-like bispecific antibodies (BsAb). Prophylaxis of hACE2 mice and post-infection treatment of golden hamsters demonstrates the efficacy of the monospecific antibodies against the original Wuhan strain, while promising in vitro results with the BsAbs demonstrate enhanced binding and distinct synergistic effects on neutralizing activity against circulating variants of concern. In particular, one BsAb engineered in a tandem scFv-Fc configuration shows synergistic neutralization activity against several variants of concern including B.1.617.2. This work provides evidence that synergistic neutralization can be achieved using a BsAb scaffold, and serves as a foundation for the future development of broadly reactive BsAbs against emerging variants of concern.

Expression and characterization of SARS-CoV-2 spike proteins.

The severe acute respiratory syndrome coronavirus 2 spike protein is a critical component of coronavirus disease 2019 vaccines and diagnostics and is also a therapeutic target. However, the spike protein is difficult to produce recombinantly because it is a large trimeric class I fusion membrane protein that is metastable and heavily glycosylated. We recently developed a prefusion-stabilized spike variant, termed HexaPro for six stabilizing proline substitutions, that can be expressed with a yield of >30 mg/L in ExpiCHO cells. This protocol describes an optimized workflow for expressing and biophysically characterizing rationally engineered spike proteins in Freestyle 293 and ExpiCHO cell lines. Although we focus on HexaPro, this protocol has been used to purify over a hundred different spike variants in our laboratories. We also provide guidance on expression quality control, long-term storage, and uses in enzyme-linked immunosorbent assays. The entire protocol, from transfection to biophysical characterization, can be completed in 7 d by researchers with basic tissue cell culture and protein purification expertise.

Structure-based design of prefusion-stabilized SARS-CoV-2 spikes.

The coronavirus disease 2019 (COVID-19) pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. We characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting higher expression than its parental construct (by a factor of 10) as well as the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A cryo-electron microscopy structure of HexaPro at a resolution of 3.2 angstroms confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes.

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion protein that is metastable and difficult to produce recombinantly in large quantities. Here, we designed and expressed over 100 structure-guided spike variants based upon a previously determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical and structural characterization of these variants identified numerous individual substitutions that increased protein yields and stability. The best variant, HexaPro, has six beneficial proline substitutions leading to ~10-fold higher expression than its parental construct and is able to withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 Å-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.

How IGF-1 activates its receptor.

The type I insulin-like growth factor receptor (IGF1R) is involved in growth and survival of normal and neoplastic cells. A ligand-dependent conformational change is thought to regulate IGF1R activity, but the nature of this change is unclear. We point out an underappreciated dimer in the crystal structure of the related Insulin Receptor (IR) with Insulin bound that allows direct comparison with unliganded IR and suggests a mechanism by which ligand regulates IR/IGF1R activity. We test this mechanism in a series of biochemical and biophysical assays and find the IGF1R ectodomain maintains an autoinhibited state in which the TMs are held apart. Ligand binding releases this constraint, allowing TM association and unleashing an intrinsic propensity of the intracellular regions to autophosphorylate. Enzymatic studies of full-length and kinase-containing fragments show phosphorylated IGF1R is fully active independent of ligand and the extracellular-TM regions. The key step triggered by ligand binding is thus autophosphorylation.

Expression of genes in cyanobacteria: adaptation of endogenous plasmids as platforms for high-level gene expression in Synechococcus sp. PCC 7002.

Synechococcus sp. PCC 7002 is an ideal model cyanobacterium for functional genomics and biotechnological applications through metabolic engineering. A gene expression system that takes advantage of its multiple, endogenous plasmids has been constructed in this cyanobacterium. The method involves the integration of foreign DNA cassettes with selectable markers into neutral sites that can be located on any of the several endogenous plasmids of this organism. We have exploited the natural transformability and powerful homologous recombination capacity of this organism by using linear DNA fragments for transformation. This approach overcomes barriers that have made the introduction and expression of foreign genes problematic in the past. Foremost among these is the natural restriction endonuclease barrier that can cleave transforming circular plasmid DNAs before they can be replicated in the cell. We describe herein the general methodology for expressing foreign and homologous genes in Synechococcus sp. PCC 7002, a comparison of several commonly used promoters, and provide examples of how this approach has successfully been used in complementation analyses and overproduction of proteins with affinity tags.