Biology subjects

Kearns, F. L.

Publications and source records attributed to Kearns, F. L..

9 recordsLinked to original sources

bioRxiv2025

An unconventional HxD motif orchestrates coatomer-dependent coronavirus morphogenesis

Assembly of infectious coronaviruses requires spike (S) protein trafficking by host coatomer, typically via a dibasic signal in the S cytoplasmic tail. However, the human embecoviruses HKU1 and OC43, as well as the model virus MHV, lack this motif. Here we identify a conserved His-x-Asp (HxD) sequence that functions as an unconventional coatomer-binding signal. Structural and biochemical analyses show that the MHV HxD motif engages coatomer subunits through distinct conformations, while cellular imaging demonstrates its role in directing S to assembly sites with the viral M-protein. Disruption of HxD-coatomer interactions impairs S incorporation and provokes compensatory viral adaptations, including emergence of a canonical dibasic motif or mutations in M-protein. Electron microscopy further reveals profound alterations in virion surface architecture. These findings uncover HxD as a previously unrecognized coatomer-targeting motif, highlighting an unexpected flexibility in coronavirus assembly pathways and broadening understanding of the cellular machinery that shapes coronavirus morphogenesis.

biochemistry↗

bioRxiv2025

SARS CoV-2 spike adopts distinct conformational ensembles in situ

Engineered recombinant Spike (S) has been invaluable for determining S structure and dynamics and is the basis for the design of most prevalent vaccines. While these vaccines have been highly efficacious for short-term protection from infection, protection waned with the emergence of variants (alpha through omicron). Here we report differences in conformational dynamics between native, membrane-embedded full-length S and recombinant S. Our virus-like particle (VLP) model mimics the native SARS CoV-2 virion by displaying S assembled with auxiliary E, M, and N proteins in a native membrane environment that captures the entirety of quaternary interactions mediated by S. Display of S on VLP obviates the requirement for stabilizing modifications that have been engineered into recombinant S for enhanced expression and solubility. Amide hydrogen/deuterium exchange mass spectrometry (HDXMS) reveals altered interprotomer contacts in VLP S trimers attributable to the presence of auxiliary proteins, membrane anchoring, and lack of engineered modifications. Our results reveal decreased dynamics in the S2 subunit and at sites spanning interprotomer contacts in VLP S with minimal differences in the N-terminal domain (NTD) and receptor binding domain (RBD). This carries implications for display of epitopes beyond NTD and RBD. In summary, despite affording efficient structural characterization, recombinant S distorts the intrinsic conformational ensemble of native S displayed on the virus surface.

biophysics↗

bioRxiv2024

Epistasis between N-terminal and receptor-binding domains drives cell entry in a bat coronavirus spike

Understanding the zoonotic risks posed by bat coronaviruses (CoVs) is critical for pandemic preparedness. Herein, we generated recombinant vesicular stomatitis viruses (rVSVs) bearing spikes from divergent bat CoVs to investigate their cell entry mechanisms. Unexpectedly, the successful recovery of rVSVs bearing the spike from SHC014, a SARS-like bat CoV, was associated with the acquisition of a novel substitution in the S2 fusion peptide-proximal region (FPPR). This substitution enhanced viral entry in both VSV and coronavirus contexts by increasing the availability of the spike receptor-binding domain to recognize its cellular receptor, ACE2. A second substitution in the spike N-terminal domain, uncovered through forward-genetic selection, interacted epistatically with the FPPR substitution to synergistically enhance spike:ACE2 interaction and viral entry. Our findings identify genetic pathways for adaptation by bat CoVs during spillover and host-to-host transmission, fitness trade-offs inherent to these pathways, and potential Achilles heels that could be targeted with countermeasures.

microbiology↗

bioRxiv2023

Allosteric modulation by the fatty acid site in the glycosylated SARS-CoV-2 spike

The trimeric spike protein plays an essential role in the SARS-CoV-2 virus lifecycle, facilitating virus entry through binding to the cellular receptor angiotensin-converting enzyme 2 (ACE2) and mediating viral-host membrane fusion. The SARS-CoV-2 spike contains a fatty acid (FA) binding site at the interface between two neighbouring receptor-binding domains. This site, also found in some other coronaviruses, binds free fatty acids such as linoleic acid. Binding at this site locks the spike in a non-infectious, closed conformation. This site is coupled to functionally important regions, but the effects of glycans on these allosteric effects have not been investigated. Understanding allostery and how this site modulates the behaviour of the spike protein could potentiate the development of promising alternative strategies for new coronavirus therapies. Here, we apply dynamical nonequilibrium molecular dynamics (D-NEMD) simulations to investigate allosteric effects of the FA site in the fully glycosylated spike of the original SARS-CoV-2 ancestral variant. The results show allosteric networks that connect the FA site to important functional regions of the protein, including some more than 40 [A] away, including the receptor binding motif, an antigenic supersite in the N-terminal domain, the furin cleavage site, regions surrounding the fusion peptide, and another allosteric site known to bind heme and biliverdin. The networks identified here highlight the complexity of the allosteric modulation in this protein and reveal a striking and unexpected connection between different allosteric sites. Notably, 65% of amino acid substitutions, deletions and insertions in the Alpha, Beta, Delta, Gamma and Omicron variants map onto or close to the identified allosteric pathways. Comparison of the FA site connections from D-NEMD in the glycosylated and non-glycosylated spikes revealed that the presence of glycans does not qualitatively change the internal allosteric pathways within the protein, with some glycans facilitating the transmission of the structural changes within and between subunits. Significance statementThe spike protein is crucial for the SARS-CoV-2 virus, enabling the fusion of the viral and host cell membranes. This protein contains several allosteric sites, including a fatty acid binding site at the interface between every two neighbouring receptor-binding domains. This site modulates the behaviour of the protein, with the binding of various free fatty acids and other small molecules influencing the spikes structure. In particular, the binding of linoleic acid, an essential fatty acid molecule, stabilizes the protein in a non-infectious locked conformation, thus making it inaccessible for binding to human receptors. Here, we investigate how the fatty acid site modulates the structural and dynamical behaviour of the fully glycosylated protein. Our work reveals complex patterns of communication between the fatty acid site and functionally important regions of the spike (including the receptor binding motif, the antigenic supersite in the N-terminal domain, the heme/biliverdin site, furin cleavage site and the fusion-peptide surrounding regions) and shed new light on the roles of glycans in this protein.

biochemistry↗

bioRxiv2023

Simulation-Driven Design of Stabilized SARS-CoV-2 Spike S2 Immunogens

The full-length prefusion-stabilized SARS-CoV-2 spike (S) is the principal antigen of COVID-19 vaccines. Vaccine efficacy has been impacted by emerging variants of concern that accumulate most of the sequence modifications in the immunodominant S1 subunit. S2, in contrast, is the most evolutionarily conserved region of the spike and can elicit broadly neutralizing and protective antibodies. Yet, S2s usage as an alternative vaccine strategy is hampered by its general instability. Here, we use a simulation-driven approach to design S2-only immunogens stabilized in a closed prefusion conformation. Molecular simulations provide a mechanistic characterization of the S2 trimers opening, informing the design of tryptophan substitutions that impart kinetic and thermodynamic stabilization. Structural characterization via cryo-EM shows the molecular basis of S2 stabilization in the closed prefusion conformation. Informed by molecular simulations and corroborated by experiments, we report an engineered S2 immunogen that exhibits increased protein expression, superior thermostability, and preserved immunogenicity against sarbecoviruses.

biophysics↗

bioRxiv2023

Glycoproteomic landscape and structural dynamics of TIM family immune checkpoints enabled by mucinase SmE

Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of biological functions. In particular, the T cell immunoglobulin and mucin-domain containing family of proteins (TIM-1, -3, -4) decorate immune cells and act as key checkpoint inhibitors in cancer. However, their dense O-glycosylation remains enigmatic both in terms of glycoproteomic landscape and structural dynamics, primarily due to the challenges associated with studying mucin domains. Here, we present a mucinase (SmE) and demonstrate its ability to selectively cleave along the mucin glycoprotein backbone, similar to others of its kind. Unlike other mucinases, though, SmE harbors the unique ability to cleave at residues bearing extremely complex glycans which enabled improved mass spectrometric analysis of several mucins, including the entire TIM family. With this information in-hand, we performed molecular dynamics (MD) simulations of TIM-3 and -4 to demonstrate how glycosylation affects structural features of these proteins. Overall, we present a powerful workflow to better understand the detailed molecular structures of the mucinome.

cancer biology↗

bioRxiv2022

Transmissible SARS-CoV-2 variants with resistance to clinical protease inhibitors

Vaccines and drugs have helped reduce disease severity and blunt the spread of SARS-CoV-2. However, ongoing virus transmission, continuous evolution, and increasing selective pressures have the potential to yield viral variants capable of resisting these interventions. Here, we investigate the susceptibility of natural variants of the main protease (Mpro/3CLpro) of SARS-CoV-2 to protease inhibitors. Multiple single amino acid changes in Mpro confer resistance to nirmatrelvir (the active component of Paxlovid). An additional clinical-stage inhibitor, ensitrelvir (Xocova), shows a different resistance mutation profile. Importantly, phylogenetic analyses indicate that several of these resistant variants have pre-existed the introduction of these drugs into the human population and are capable of spreading. These results encourage the monitoring of resistance variants and the development of additional protease inhibitors and other antiviral drugs with different mechanisms of action and resistance profiles for combinatorial therapy. One Sentence SummaryResistance to protease inhibitor drugs, nirmatrelvir (Paxlovid) and ensitrelvir (Xocova), exists in SARS-CoV-2 variants in the human population.

microbiology↗

bioRxiv2022

Targeted protein S-nitrosylation of ACE2 as potential treatment to prevent spread of SARS-CoV-2 infection

Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.

microbiology↗

bioRxiv2021

A glycan gate controls opening of the SARS-CoV-2 spike protein

SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded "down" to an exposed "up" state in order to bind the human ACE2 receptor and infect cells. While snapshots of the "up" and "down" states have been obtained by cryoEM and cryoET, details of the RBD opening transition evade experimental characterization. Here, over 130 s of weighted ensemble (WE) simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD opening pathways. Together with ManifoldEM analysis of cryo-EM data and biolayer interferometry experiments, we reveal a gating role for the N-glycan at position N343, which facilitates RBD opening. Residues D405, R408, and D427 also participate. The atomic-level characterization of the glycosylated spike activation mechanism provided herein achieves a new high-water mark for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection.

biophysics↗