Biology subjects

King, N. P.

Publications and source records attributed to King, N. P..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Solubilization of Membrane Proteins using designed protein WRAPS

The development of therapies and vaccines targeting integral membrane proteins has been complicated by their extensive hydrophobic surfaces, which can make production and structural characterization difficult. Here we describe a general deep learning-based design approach for solubilizing native membrane proteins while preserving their sequence, fold, and function using genetically encoded de novo protein WRAPs (Water-soluble RFdiffused Amphipathic Proteins) that surround the lipid-interacting hydrophobic surfaces, rendering them stable and water-soluble without the need for detergents. We design WRAPs for both beta-barrel outer membrane and helical multi-pass transmembrane proteins, and show that the solubilized proteins retain the binding and enzymatic functions of the native targets with enhanced stability. Syphilis vaccine development has been hindered by difficulties in characterizing and producing the outer membrane protein antigens; we generated soluble versions of four Treponema pallidum outer membrane beta barrels which are potential syphilis vaccine antigens. A 4.0 [A] cryo-EM map of WRAPed TP0698 is closely consistent with the design model. WRAPs should be broadly useful for facilitating biochemical and structural characterization of integral membrane proteins, enabling therapeutic discovery by screening against purified soluble targets, and generating antigenically intact immunogens for vaccine development.

biochemistry↗

bioRxiv2025

A Multivalent Pan-Ebolavirus Nanoparticle Vaccine Provides Protection in Rodents from Lethal Infection by Adapted Zaire and Sudan Viruses

Both Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV) are members of the family Filoviridae, first discovered in 1976 during outbreaks of hemorrhagic fever in northern Zaire and southern Sudan. Ebola virus disease outbreaks are major public health events because of their potential for human-to-human transmission with high case fatality rates. Filoviral surface glycoproteins (GPs) are known to be the primary targets of neutralizing antibodies for protection from disease, and are the relevant immunogens in the two approved EBOV vaccines. Here we describe the design, electron microscopy-based structural characterization, and efficacy testing of a series of icosahedral I53-50 nanoparticles displaying prefusion trimeric EBOV and SUDV GP antigens. Mice and guinea pigs vaccinated with either a cocktail of EBOV-GP-I53-50 plus SUDV-GP-I53-50 or mosaic EBOV / SUDV-GP-I53-50 nanoparticles were protected from death or severe clinical signs of disease and weight loss, respectively, when challenged with either mouse-adapted EBOV or guinea pig-adapted SUDV.

immunology↗

bioRxiv2024

Deep mutational scanning of rabies glycoprotein defines mutational constraint and antibody-escape mutations

Rabies virus causes nearly 60,000 human deaths annually. Antibodies that target the rabies glycoprotein (G) are being developed as post-exposure prophylactics, but mutations in G can render such antibodies ineffective. Here, we use pseudovirus deep mutational scanning to measure how all single amino-acid mutations to G affect cell entry and neutralization by a panel of antibodies. These measurements identify sites critical for rabies Gs function, and define constrained regions that are attractive epitopes for clinical antibodies, including at the apex and base of the protein. We provide complete maps of escape mutations for eight monoclonal antibodies, including some in clinical use or development. Escape mutations for most antibodies are present in some natural rabies strains. Overall, this work provides comprehensive information on the functional and antigenic effects of G mutations that can help inform development of stabilized vaccine antigens and antibodies that are resilient to rabies genetic variation.

microbiology↗

bioRxiv2024

Potent antitumor activity of a designed interleukin-21 mimic

Long-standing goals of cancer immunotherapy are to activate cytotoxic antitumor T cells across a broad range of affinities while dampening suppressive regulatory T (Treg) cell responses, but current approaches achieve these goals with limited success. Here, we report a de novo IL-21 mimic, 21h10, designed to have augmented stability and high signaling potency in both humans and mice. In multiple animal models and in ex vivo human melanoma patient derived organotypic tumor spheroids (PDOTS), 21h10 showed robust antitumor activity. 21h10 generates significantly prolonged STAT signaling in vivo compared with native IL-21, and has considerably stronger anti-tumor activity. Toxicities associated with systemic administration of 21h10 could be mitigated by TNF blockade without compromising antitumor efficacy. In the tumor microenvironment, 21h10 induced highly cytotoxic antitumor T cells from clonotypes with a range of affinities for endogenous tumor antigens, robustly expanding low-affinity cytotoxic T cells and driving high expression of interferon- (IFN-) and granzyme B compared to native IL-21, while increasing the frequency of IFN-+ Th1 cells and reducing that of Foxp3+ Tregs. As 21h10 has full human/mouse cross-reactivity, high stability and potency, and potentiates low-affinity antitumor responses, it has considerable translational potential.

biochemistry↗

bioRxiv2024

Structural Convergence and Water-Mediated Substrate Mimicry Enable Broad Neuraminidase Inhibition by Human Antibodies

Influenza has been responsible for multiple global pandemics and seasonal epidemics and claimed millions of lives. The imminent threat of a panzootic outbreak of avian influenza H5N1 virus underscores the urgent need for pandemic preparedness and effective countermeasures, including monoclonal antibodies (mAbs). Here, we characterize human mAbs that target the highly conserved catalytic site of viral neuraminidase (NA), termed NCS mAbs, and the molecular basis of their broad specificity. Cross-reactive NA-specific B cells were isolated by using stabilized NA probes of non-circulating subtypes. We found that NCS mAbs recognized multiple NAs of influenza A as well as influenza B NAs and conferred prophylactic protections in mice against H1N1, H5N1, and influenza B viruses. Cryo-electron microscopy structures of two NCS mAbs revealed that they rely on structural mimicry of sialic acid, the substrate of NA, by coordinating not only amino acid side chains but also water molecules, enabling inhibition of NA activity across multiple influenza A and B viruses, including avian influenza H5N1 clade 2.3.4.4b viruses. Our results provide a molecular basis for the broad reactivity and inhibitory activity of NCS mAbs targeting the catalytic site of NA through substrate mimicry.

immunology↗

bioRxiv2024

Computational design of bifaceted protein nanomaterials with tailorable properties

Recent advances in computational methods have led to considerable progress in the design of self-assembling protein nanoparticles. However, nearly all nanoparticles designed to date exhibit strict point group symmetry, with each subunit occupying an identical, symmetrically related environment. This limits the structural diversity that can be achieved and precludes anisotropic functionalization. Here, we describe a general computational strategy for designing multi-component bifaceted protein nanomaterials with two distinctly addressable sides. The method centers on docking pseudosymmetric heterooligomeric building blocks in architectures with dihedral symmetry and designing an asymmetric protein-protein interface between them. We used this approach to obtain an initial 30-subunit assembly with pseudo-D5 symmetry, and then generated an additional 15 variants in which we controllably altered the size and morphology of the bifaceted nanoparticles by designing de novo extensions to one of the subunits. Functionalization of the two distinct faces of the nanoparticles with de novo protein minibinders enabled specific colocalization of two populations of polystyrene microparticles coated with target protein receptors. The ability to accurately design anisotropic protein nanomaterials with precisely tunable structures and functions could be broadly useful in applications that require colocalizing two or more distinct target moieties.

bioengineering↗

bioRxiv2024

Computational redesign of TALE proteins for DNA-templated assembly of protein fibers

Many viral proteins self-assemble into capsid structures, often using their genetic material as a template for assembly. To date, de novo designed capsid-like proteins do not require genetic material as a template for assembly, which can be both an advantage and a disadvantage depending on the use case. Templates are indispensable, for example, in the assembly of linear structures with well-defined lengths. As a first step towards fully de novo designed templated assembly, here we redesign proteins from the Transcription activator-like effector (TALE) family of transcriptional regulators to polymerize on double-stranded DNA (dsDNA) templates. Starting from natural TALE protein sequences, we created idealized repeat proteins with sequence-independent DNA binding properties that cooperatively self-assemble to form linear protein-DNA complexes with template-controlled lengths. We used high-resolution atomic force microscopy (AFM) and cryo electron microscopy (cryo-EM) to characterize the three-dimensional structures of the DNA-protein hybrid complexes. In these structures, a protein filament helically wraps around the dsDNA using a binding mode similar to that of natural TALE proteins. As an example application of these materials, we show the system can be used for repetitive peptide antigen display at precisely controlled repeat distances, and that such immunogens elicit robust antigen-specific antibodies in mice.

biochemistry↗

bioRxiv2024

Protein identification using cryo-EM and artificial intelligence guides improved sample purification

Protein purification is essential in protein biochemistry, structural biology, and protein design. It enables the determination of protein structures, the study of biological mechanisms, and the biochemical and biophysical characterization of both natural and de novo designed proteins. Despite the broad application of various protein purification protocols, standard strategies can still encounter challenges, such as the unintended co-purification of unknown contaminants alongside the target protein. In particular, co-purification issues pose significant challenges for designed self-assembling protein nanomaterials, as it is difficult to determine whether unexpected observed geometries represent novel assembly states of the designed system, cross-contamination from other assemblies, or native proteins originating from the expression host. In this study, we assessed the ability of an automated structure-to-sequence pipeline to unambiguously identify an unknown co-purifying protein found across several purified designed protein samples. Using cryo-electron microscopy (Cryo-EM), ModelAngelos sequence-agnostic automated model-building feature, and the Basic Local Alignment Search Tool (BLAST), we identified the unknown protein as dihydrolipoamide succinyltransferase (DLST). This identification was further confirmed by comparing the cryo-EM data with available DLST structures in the Protein Data Bank (PDB) and AlphaFold 3 predictions from the top BLAST hits. The clear identification of DLST informed our subsequent literature search and led to the rational modification of our protein purification protocol, ultimately enabling the exclusion of the contaminant from preparations of our target nanoparticle. This study demonstrates the successful application of a structure-to-sequence workflow, integrating Cryo-EM, ModelAngelo, protein BLAST, PDB structures, and AlphaFold 3 predictions, to identify and remove an unknown protein from a purified sample. It also highlights the broader potential of integrating Cryo-EM with AI-driven tools for accurate protein identification across various samples and contexts within protein science. HighlightsO_LIAn unknown protein was consistently found in multiple de novo designed protein samples. C_LIO_LIThe protein was identified as dihydrolipoamide succinyltransferase (DLST) using Cryo-EM, ModelAngelo and BLAST, and further verified using AlphaFold 3 and the PDB. C_LIO_LIIdentification enabled rational modification of the purification protocol to exclude the contaminant. C_LIO_LIThis method enables accurate protein identification without requiring near-atomic resolution or prior sequence and structural data, making it broadly applicable to various areas of protein science. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/612515v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@592410org.highwire.dtl.DTLVardef@4ea3f3org.highwire.dtl.DTLVardef@edd0bcorg.highwire.dtl.DTLVardef@122fba2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Molecular basis of pathogenicity of the recently emerged FCoV-23 coronavirus

The ability of coronaviruses to recombine and cross species barriers affects human and animal health globally and is a pandemic threat. FCoV-23 is a recently emerged, highly pathogenic recombinant coronavirus responsible for a widespread outbreak of feline infectious peritonitis (FIP) likely linked to in-host viral evolution. Here, we report cryoEM structures of two FCoV-23 spike (S) isoforms explaining that the in-host loss of domain 0 observed in clinical samples enhances entry into cells and fusogenicity by facilitating protease access, leading to biotype switching and lethality. We show that FCoV-23 can use several aminopeptidase N (APN) orthologs as receptors and reveal the molecular determinants of receptor species tropism, including a glycan modulating human receptor utilization. We define antigenic relationships among alphacoronaviruses infecting humans and other mammalian species and identify a cross-reactive alphacoronavirus monoclonal antibody inhibiting FCoV-23 pseudovirus entry, paving the way for vaccine and therapeutic development targeting this highly pathogenic virus.

biochemistry↗

bioRxiv2024

Computationally designed mRNA-launched protein nanoparticle vaccines

Both protein nanoparticle and mRNA vaccines were clinically de-risked during the COVID-19 pandemic1-6. These vaccine modalities have complementary strengths: antigen display on protein nanoparticles can enhance the magnitude, quality, and durability of antibody responses7-10, while mRNA vaccines can be rapidly manufactured11 and elicit antigen-specific CD4 and CD8 T cells12,13. Here we leverage a computationally designed icosahedral protein nanoparticle that was redesigned for optimal secretion from eukaryotic cells14 to develop an mRNA-launched nanoparticle vaccine for SARS-CoV-2. The nanoparticle, which displays 60 copies of a stabilized variant of the Wuhan-Hu-1 Spike receptor binding domain (RBD)15, formed monodisperse, antigenically intact assemblies upon secretion from transfected cells. An mRNA vaccine encoding the secreted RBD nanoparticle elicited 5- to 28-fold higher levels of neutralizing antibodies than an mRNA vaccine encoding membrane-anchored Spike, induced higher levels of CD8 T cells than the same immunogen when delivered as an adjuvanted protein nanoparticle, and protected mice from vaccine-matched and -mismatched SARS-CoV-2 challenge. Our data establish that delivering protein nanoparticle immunogens via mRNA vaccines can combine the benefits of each modality and, more broadly, highlight the utility of computational protein design in genetic immunization strategies.

immunology↗

bioRxiv2024

Deep mutational scanning of H5 hemagglutinin to inform influenza virus surveillance

H5 influenza is a potential pandemic threat. Previous studies have identified molecular phenotypes of the viral hemagglutinin (HA) protein that contribute to pandemic risk, including cell entry, receptor preference, HA stability, and reduced neutralization by polyclonal sera. Here we use pseudovirus deep mutational scanning to measure how all mutations to a clade 2.3.4.4b H5 HA affect each phenotype. We identify mutations that allow HA to better bind 2-6-linked sialic acids, and show that some viruses already carry mutations that stabilize HA. We also identify recent viral strains with reduced neutralization to sera elicited by candidate vaccine virus. Overall, the systematic nature of deep mutational scanning combined with the safety of pseudoviruses enables comprehensive characterization of mutations to inform surveillance of H5 influenza.

microbiology↗

bioRxiv2024

De novo design of protein minibinder agonists of TLR3

Toll-like Receptor 3 (TLR3) is a pattern recognition receptor that initiates antiviral immune responses upon binding double-stranded RNA (dsRNA). Several nucleic acid-based TLR3 agonists have been explored clinically as vaccine adjuvants in cancer and infectious disease, but present substantial manufacturing and formulation challenges. Here, we use computational protein design to create novel miniproteins that bind to human TLR3 with nanomolar affinities. Cryo-EM structures of two minibinders in complex with TLR3 reveal that they bind the target as designed, although one partially unfolds due to steric competition with a nearby N-linked glycan. Multimeric forms of both minibinders induce NF-{kappa}B signaling in TLR3-expressing cell lines, demonstrating that they may have therapeutically relevant biological activity. Our work provides a foundation for the development of specific, stable, and easy-to-formulate protein-based agonists of TLRs and other pattern recognition receptors.

biochemistry↗

bioRxiv2024

Protein nanoparticle vaccines induce potent neutralizing antibody responses against MERS-CoV

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that causes severe and often lethal respiratory illness in humans. The MERS-CoV spike (S) protein is the viral fusogen and the target of neutralizing antibodies, and has therefore been the focus of vaccine design efforts. Currently there are no licensed vaccines against MERS-CoV and only a few candidates have advanced to Phase I clinical trials. Here we developed MERS-CoV vaccines utilizing a computationally designed protein nanoparticle platform that has generated safe and immunogenic vaccines against various enveloped viruses, including a licensed vaccine for SARS-CoV-2. Two-component protein nanoparticles displaying MERS-CoV S-derived antigens induced robust neutralizing antibody responses and protected mice against challenge with mouse-adapted MERS-CoV. Electron microscopy polyclonal epitope mapping and serum competition assays revealed the specificities of the dominant antibody responses elicited by immunogens displaying the prefusion-stabilized S-2P trimer, receptor binding domain (RBD), or N-terminal domain (NTD). An RBD nanoparticle vaccine elicited antibodies targeting multiple non-overlapping epitopes in the RBD, whereas anti-NTD antibodies elicited by the S-2P- and NTD-based immunogens converged on a single antigenic site. Our findings demonstrate the potential of two-component nanoparticle vaccine candidates for MERS-CoV and suggest that this platform technology could be broadly applicable to betacoronavirus vaccine development.

immunology↗

bioRxiv2024

Deep mutational scanning reveals functional constraints and antigenic variability of Lassa virus glycoprotein complex

Lassa virus is estimated to cause thousands of human deaths per year, primarily due to spillovers from its natural host, Mastomys rodents. Efforts to create vaccines and antibody therapeutics must account for the evolutionary variability of Lassa viruss glycoprotein complex (GPC), which mediates viral entry into cells and is the target of neutralizing antibodies. To map the evolutionary space accessible to GPC, we use pseudovirus deep mutational scanning to measure how nearly all GPC amino-acid mutations affect cell entry and antibody neutralization. Our experiments define functional constraints throughout GPC. We quantify how GPC mutations affect neutralization by a panel of monoclonal antibodies and show that all antibodies are escaped by mutations that exist among natural Lassa virus lineages. Overall, our work describes a biosafety-level-2 method to elucidate the mutational space accessible to GPC and shows how prospective characterization of antigenic variation could aid design of therapeutics and vaccines.

microbiology↗

bioRxiv2023

A broadly generalizable stabilization strategy for sarbecovirus fusion machinery vaccines

Continuous evolution of SARS-CoV-2 alters the antigenicity of the immunodominant spike (S) receptor-binding domain and N-terminal domain, undermining the efficacy of vaccines and monoclonal antibody therapies. To overcome this challenge, we set out to develop a vaccine focusing antibody responses on the highly conserved but metastable S2 subunit, which folds as a spring-loaded fusion machinery. Here, we describe a protein design strategy enabling prefusion-stabilization of the SARS-CoV-2 S2 subunit and high yield recombinant expression of trimers with native structure and antigenicity. We demonstrate that our design strategy is broadly generalizable to all sarbecoviruses, as exemplified with the SARS-CoV-1 (clade 1a) and PRD-0038 (clade 3) S2 fusion machineries. Immunization of mice with a prefusion-stabilized SARS-CoV-2 S2 trimer vaccine elicits broadly reactive sarbecovirus antibody responses and neutralizing antibody titers of comparable magnitude against Wuhan-Hu-1 and the immune evasive XBB.1.5 variant. Vaccinated mice were protected from weight loss and disease upon challenge with SARS-CoV-2 XBB.1.5, providing proof-of-principle for fusion machinery sarbecovirus vaccines motivating future development.

biochemistry↗

bioRxiv2023

Local structural flexibility drives oligomorphism in computationally designed protein assemblies

Many naturally occurring protein assemblies have dynamic structures that allow them to perform specialized functions. For example, clathrin coats adopt a wide variety of architectures to adapt to vesicular cargos of various sizes. Although computational methods for designing novel self-assembling proteins have advanced substantially over the past decade, most existing methods focus on designing static structures with high accuracy. Here we characterize the structures of three distinct computationally designed protein assemblies that each form multiple unanticipated architectures, and identify flexibility in specific regions of the subunits of each assembly as the source of structural diversity. Cryo-EM single-particle reconstructions and native mass spectrometry showed that only two distinct architectures were observed in two of the three cases, while we obtained six cryo-EM reconstructions that likely represent a subset of the architectures present in solution in the third case. Structural modeling and molecular dynamics simulations indicated that the surprising observation of a defined range of architectures, instead of non-specific aggregation, can be explained by constrained flexibility within the building blocks. Our results suggest that deliberate use of structural flexibility as a design principle will allow exploration of previously inaccessible structural and functional space in designed protein assemblies.

bioengineering↗

bioRxiv2023

Broad receptor tropism and immunogenicity of a clade 3 sarbecovirus

Although Rhinolophus bats harbor diverse clade 3 sarbecoviruses, the structural determinants of receptor tropism along with the antigenicity of their spike (S) glycoproteins remain uncharacterized. Here, we show that the African Rinolophus bat clade 3 sarbecovirus PRD-0038 S has a broad ACE2 usage and that RBD mutations further expand receptor promiscuity and enable human ACE2 utilization. We determined a cryoEM structure of the PRD-0038 RBD bound to R. alcyone ACE2, explaining receptor tropism and highlighting differences with SARS-CoV-1 and SARS-CoV-2. Characterization of PRD-0038 S using cryoEM and monoclonal antibody reactivity revealed its distinct antigenicity relative to SARS-CoV-2 and identified PRD-0038 cross-neutralizing antibodies for pandemic preparedness. PRD-0038 S vaccination elicited greater titers of antibodies cross-reacting with vaccine-mismatched clade 2 and clade 1a sarbecoviruses compared to SARS-CoV-2 S due to broader antigenic targeting, motivating the inclusion of clade 3 antigens in next-generation vaccines for enhanced resilience to viral evolution.

biochemistry↗

bioRxiv2023

Rapid and automated design of two-component protein nanomaterials using ProteinMPNN

The design of novel protein-protein interfaces using physics-based design methods such as Rosetta requires substantial computational resources and manual refinement by expert structural biologists. A new generation of deep learning methods promises to simplify protein-protein interface design and enable its application to a wide variety of problems by researchers from various scientific disciplines. Here we test the ability of a deep learning method for protein sequence design, ProteinMPNN, to design two-component tetrahedral protein nanomaterials and benchmark its performance against Rosetta. ProteinMPNN had a similar success rate to Rosetta, yielding 13 new experimentally confirmed assemblies, but required orders of magnitude less computation and no manual refinement. The interfaces designed by ProteinMPNN were substantially more polar than those designed by Rosetta, which facilitated in vitro assembly of the designed nanomaterials from independently purified components. Crystal structures of several of the assemblies confirmed the accuracy of the design method at high resolution. Our results showcase the potential of deep learning-based methods to unlock the widespread application of designed protein-protein interfaces and self-assembling protein nanomaterials in biotechnology.

biochemistry↗