Rapid simulation of glycoprotein structures by grafting and steric exclusion of glycan conformer libraries.

Most membrane proteins are modified by covalent addition of complex sugars through N- and O-glycosylation. Unlike proteins, glycans do not typically adopt specific secondary structures and remain very mobile, shielding potentially large fractions of protein surface. High glycan conformational freedom hinders complete structural elucidation of glycoproteins. Computer simulations may be used to model glycosylated proteins but require hundreds of thousands of computing hours on supercomputers, thus limiting routine use. Here, we describe GlycoSHIELD, a reductionist method that can be implemented on personal computers to graft realistic ensembles of glycan conformers onto static protein structures in minutes. Using molecular dynamics simulation, small-angle X-ray scattering, cryoelectron microscopy, and mass spectrometry, we show that this open-access toolkit provides enhanced models of glycoprotein structures. Focusing on N-cadherin, human coronavirus spike proteins, and gamma-aminobutyric acid receptors, we show that GlycoSHIELD can shed light on the impact of glycans on the conformation and activity of complex glycoproteins.

Sialic acid-containing glycolipids mediate binding and viral entry of SARS-CoV-2.

Emerging evidence suggests that host glycans influence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we reveal that the receptor-binding domain (RBD) of the spike (S) protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (Sia), with preference for monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind to the RBD. The monomeric affinities (Kd = 100-200 µM) of gangliosides for the RBD are similar to another negatively charged glycan ligand of the RBD proposed as a viral co-receptor, heparan sulfate (HS) dp2-dp6 oligosaccharides. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to angiotensin-converting enzyme 2 (ACE2)-expressing cells is decreased following depletion of cell surface Sia levels using three approaches: sialyltransferase (ST) inhibition, genetic knockout of Sia biosynthesis, or neuraminidase treatment. These effects on RBD binding and both pseudotyped and authentic SARS-CoV-2 viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.

An N-glycopeptide MS/MS data analysis workflow leveraging two complementary glycoproteomic software tools for more confident identification and assignments.

Complete coverage of all N-glycosylation sites on the SARS-CoV2 spike protein would require the use of multiple proteases in addition to trypsin. Subsequent identification of the resulting glycopeptides by searching against database often introduces assignment errors due to similar mass differences between different permutations of amino acids and glycosyl residues. By manually interpreting the individual MS2 spectra, we report here the common sources of errors in assignment, especially those introduced by the use of chymotrypsin. We show that by applying a stringent threshold of acceptance, erroneous assignment by the commonly used Byonic software can be controlled within 15%, which can be reduced further if only those also confidently identified by a different search engine, pGlyco3, were considered. A representative site-specific N-glycosylation pattern could be constructed based on quantifying only the overlapping subset of N-glycopeptides identified at higher confidence. Applying the two complimentary glycoproteomic software in a concerted data analysis workflow, we found and confirmed that glycosylation at several sites of an unstable Omicron spike protein differed significantly from those of the stable trimeric product of the parental D614G variant.

Structure-guided antibody cocktail for prevention and treatment of COVID-19.

Development of effective therapeutics for mitigating the COVID-19 pandemic is a pressing global need. Neutralizing antibodies are known to be effective antivirals, as they can be rapidly deployed to prevent disease progression and can accelerate patient recovery without the need for fully developed host immunity. Here, we report the generation and characterization of a series of chimeric antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Some of these antibodies exhibit exceptionally potent neutralization activities in vitro and in vivo, and the most potent of our antibodies target three distinct non-overlapping epitopes within the RBD. Cryo-electron microscopy analyses of two highly potent antibodies in complex with the SARS-CoV-2 spike protein suggested they may be particularly useful when combined in a cocktail therapy. The efficacy of this antibody cocktail was confirmed in SARS-CoV-2-infected mouse and hamster models as prophylactic and post-infection treatments. With the emergence of more contagious variants of SARS-CoV-2, cocktail antibody therapies hold great promise to control disease and prevent drug resistance.

Cryo-EM analysis of a feline coronavirus spike protein reveals a unique structure and camouflaging glycans.

Feline infectious peritonitis virus (FIPV) is an alphacoronavirus that causes a nearly 100% mortality rate without effective treatment. Here we report a 3.3-Å cryoelectron microscopy (cryo-EM) structure of the serotype I FIPV spike (S) protein, which is responsible for host recognition and viral entry. Mass spectrometry provided site-specific compositions of densely distributed high-mannose and complex-type N-glycans that account for 1/4 of the total molecular mass; most of the N-glycans could be visualized by cryo-EM. Specifically, the N-glycans that wedge between 2 galectin-like domains within the S1 subunit of FIPV S protein result in a unique propeller-like conformation, underscoring the importance of glycosylation in maintaining protein structures. The cleavage site within the S2 subunit responsible for activation also showed distinct structural features and glycosylation. These structural insights provide a blueprint for a better molecular understanding of the pathogenesis of FIP.

D614G mutation in the SARS-CoV-2 spike protein enhances viral fitness by desensitizing it to temperature-dependent denaturation.

The D614G mutation in the spike protein of SARS-CoV-2 alters the fitness of the virus, leading to the dominant form observed in the COVID-19 pandemic. However, the molecular basis of the mechanism by which this mutation enhances fitness is not clear. Here we demonstrated by cryo-electron microscopy that the D614G mutation resulted in increased propensity of multiple receptor-binding domains (RBDs) in an upward conformation poised for host receptor binding. Multiple substates within the one RBD-up or two RBD-up conformational space were determined. According to negative staining electron microscopy, differential scanning calorimetry, and differential scanning fluorimetry, the most significant impact of the mutation lies in its ability to eliminate the unusual cold-induced unfolding characteristics and to significantly increase the thermal stability under physiological pH. The D614G spike variant also exhibited exceptional long-term stability when stored at 37 °C for up to 2 months. Our findings shed light on how the D614G mutation enhances the infectivity of SARS-CoV-2 through a stabilizing mutation and suggest an approach for better design of spike protein-based conjugates for vaccine development.

Distinct shifts in site-specific glycosylation pattern of SARS-CoV-2 spike proteins associated with arising mutations in the D614G and Alpha variants.

Extensive glycosylation of the spike protein of severe acute respiratory syndrome coronavirus 2 virus not only shields the major part of it from host immune responses, but glycans at specific sites also act on its conformation dynamics and contribute to efficient host receptor binding, and hence infectivity. As variants of concern arise during the course of the coronavirus disease of 2019 pandemic, it is unclear if mutations accumulated within the spike protein would affect its site-specific glycosylation pattern. The Alpha variant derived from the D614G lineage is distinguished from others by having deletion mutations located right within an immunogenic supersite of the spike N-terminal domain (NTD) that make it refractory to most neutralizing antibodies directed against this domain. Despite maintaining an overall similar structural conformation, our mass spectrometry-based site-specific glycosylation analyses of similarly produced spike proteins with and without the D614G and Alpha variant mutations reveal a significant shift in the processing state of N-glycans on one specific NTD site. Its conversion to a higher proportion of complex type structures is indicative of altered spatial accessibility attributable to mutations specific to the Alpha variant that may impact its transmissibility. This and other more subtle changes in glycosylation features detected at other sites provide crucial missing information otherwise not apparent in the available cryogenic electron microscopy-derived structures of the spike protein variants.

A booster dose of Delta × Omicron hybrid mRNA vaccine produced broadly neutralizing antibody against Omicron and other SARS-CoV-2 variants.

BACKGROUND: With the continuous emergence of new SARS-CoV-2 variants that feature increased transmission and immune escape, there is an urgent demand for a better vaccine design that will provide broader neutralizing efficacy. METHODS: We report an mRNA-based vaccine using an engineered "hybrid" receptor binding domain (RBD) that contains all 16 point-mutations shown in the currently prevailing Omicron and Delta variants. RESULTS: A booster dose of hybrid vaccine in mice previously immunized with wild-type RBD vaccine induced high titers of broadly neutralizing antibodies against all tested SARS-CoV-2 variants of concern (VOCs). In naïve mice, hybrid vaccine generated strong Omicron-specific neutralizing antibodies as well as low but significant titers against other VOCs. Hybrid vaccine also elicited CD8+/IFN-γ+ T cell responses against a conserved T cell epitope present in wild type and all VOCs. CONCLUSIONS: These results demonstrate that inclusion of different antigenic mutations from various SARS-CoV-2 variants is a feasible approach to develop cross-protective vaccines.

Direct Visualization of a 26 kDa Protein by Cryo-Electron Microscopy Aided by a Small Scaffold Protein.

Cryo-electron microscopy (cryo-EM)-based structure determination of small proteins is hindered by the technical challenges associated with low signal-to-noise ratios of their particle images in intrinsically noisy micrographs. One solution is to attach the target protein to a large protein scaffold to increase its apparent size and, therefore, image contrast. Here we report a novel scaffold design based on a trimeric helical protein, E. coli ornithine transcarbamylase (OTC), fused to human ubiquitin. As a proof of principle, we demonstrated the ability to resolve a cryo-EM map of a 26 kDa human ubiquitin C-terminal hydrolase (UCHL1) attached to the C-terminus of ubiquitin as part of the trimeric assembly. The results revealed conformational changes in UCHL1 upon binding to ubiquitin, namely, a significant displacement of α-helix 2, which was also observed by X-ray crystallography. Our findings demonstrated the potential of the trimeric OTC scaffold design for studying a large number of ubiquitin interacting proteins by cryo-EM.

Comparative folding analyses of unknotted versus trefoil-knotted ornithine transcarbamylases suggest stabilizing effects of protein knots.

Ornithine transcarbamylases (OTCs) are conserved enzymes involved in arginine biosynthesis in microbes and the urea cycle in mammals. Recent bioinformatics analyses identified two unique OTC variants, N-succinyl-l-ornithine transcarbamylase from Bacteroides fragilis (BfSOTC) and N-acetyl-l-ornithine transcarbamylase from Xanthomonas campestris (XcAOTC). These two variants diverged from other OTCs during evolution despite sharing the common tertiary and quaternary structures, with the exception that the substrate recognition motifs are topologically knotted. The OTC family therefore offers a unique opportunity for investigating the importance of protein knots in biological functions and folding stabilities. Using hydrogen-deuterium exchange-coupled mass spectrometry, we compared the native dynamics of BfSOTC and XcAOTC with respect to the unknotted ornithine transcarbamylase from Escherichia coli (EcOTC). Our results suggest that, in addition to substrate specificity, the knotted structures in XcAOTC and BfSOTC may play an important role in stabilizing the folding dynamics, particularly around the knotted structural elements.

Biophysical and structural characterizations of the effects of mutations on the structure-activity relationships of SARS-CoV-2 spike protein.

Mutations on the spike (S) protein of SARS-CoV-2 could induce structural changes that help increase viral transmissibility and enhance resistance to antibody neutralization. Here, we report a robust workflow to prepare recombinant S protein variants and its host receptor angiotensin-convert enzyme 2 (ACE2) by using a mammalian cell expression system. The functional states of the S protein variants are investigated by cryo-electron microscopy (cryo-EM) and negative staining electron microscopy (NSEM) to visualize their molecular structures in response to mutations, receptor binding, antibody binding, and environmental changes. The folding stabilities of the S protein variants can be deduced from morphological changes based on NSEM imaging analysis. Differential scanning calorimetry provides thermodynamic information to complement NSEM. Impacts of the mutations on host receptor binding and antibody neutralization are in vitro by kinetic binding analyses in addition to atomic insights gleaned from cryo-electron microscopy (cryo-EM). This experimental strategy is generally applicable to studying the molecular basis of host-pathogen interactions.

Tumor suppressor BAP1 nuclear import is governed by transportin-1.

Subcellular localization of the deubiquitinating enzyme BAP1 is deterministic for its tumor suppressor activity. While the monoubiquitination of BAP1 by an atypical E2/E3-conjugated enzyme UBE2O and BAP1 auto-deubiquitination are known to regulate its nuclear localization, the molecular mechanism by which BAP1 is imported into the nucleus has remained elusive. Here, we demonstrated that transportin-1 (TNPO1, also known as Karyopherin ß2 or Kapß2) targets an atypical C-terminal proline-tyrosine nuclear localization signal (PY-NLS) motif of BAP1 and serves as the primary nuclear transporter of BAP1 to achieve its nuclear import. TNPO1 binding dissociates dimeric BAP1 and sequesters the monoubiquitination sites flanking the PY-NLS of BAP1 to counteract the function of UBE2O that retains BAP1 in the cytosol. Our findings shed light on how TNPO1 regulates the nuclear import, self-association, and monoubiquitination of BAP1 pertinent to oncogenesis.

Impacts of Cancer-associated Mutations on the Structure-Activity Relationship of BAP1.

BRAC1 associated protein-1 (BAP1) is a major tumor suppressor involved in many cancers. The deubiquitinase (DUB) activity of BAP1 is essential for its nuclear localization, histone remodeling and proteostasis associated with mitochondrial calcium flux. Loss of the DUB activity due to catalytic mutations within the ubiquitin C-terminal hydrolase (UCH) domain of BAP1 (BAP1-UCH) directly contributes to oncogenesis. Nevertheless, it is non-trivial to rationalize how the other high-frequency but non-catalytic mutations within the BAP1-UCH lead to malignancies. Here we used multiplex spectroscopic, thermodynamic and biophysical analyses to investigate the impacts of eleven high-occurrence mutations within BAP1-UCH on the structure, folding and function. Several mutations significantly destabilize BAP1-UCH and increase its aggregation propensity. Hydrogen-deuterium exchange mass spectrometry data revealed allosteric destabilizations caused by mutations distant from the catalytic site. Our findings gave a comprehensive and multiscale account of the molecular basis of how these non-catalytic mutations within BAP1-UCH may be implicated in oncogenesis.

In situ structure and dynamics of an alphacoronavirus spike protein by cryo-ET and cryo-EM.

Porcine epidemic diarrhea (PED) is a highly contagious swine disease caused by porcine epidemic diarrhea virus (PEDV). PED causes enteric disorders with an exceptionally high fatality in neonates, bringing substantial economic losses in the pork industry. The trimeric spike (S) glycoprotein of PEDV is responsible for virus-host recognition, membrane fusion, and is the main target for vaccine development and antigenic analysis. The atomic structures of the recombinant PEDV S proteins of two different strains have been reported, but they reveal distinct N-terminal domain 0 (D0) architectures that may correspond to different functional states. The existence of the D0 is a unique feature of alphacoronavirus. Here we combined cryo-electron tomography (cryo-ET) and cryo-electron microscopy (cryo-EM) to demonstrate in situ the asynchronous S protein D0 motions on intact viral particles of a highly virulent PEDV Pintung 52 strain. We further determined the cryo-EM structure of the recombinant S protein derived from a porcine cell line, which revealed additional domain motions likely associated with receptor binding. By integrating mass spectrometry and cryo-EM, we delineated the complex compositions and spatial distribution of the PEDV S protein N-glycans, and demonstrated the functional role of a key N-glycan in modulating the D0 conformation.

Phylogenetic and Structural Analysis of NIN-Like Proteins With a Type I/II PB1 Domain That Regulates Oligomerization for Nitrate Response.

Absorption of macronutrients such as nitrogen is a critical process for land plants. There is little information available on the correlation between the root evolution of land plants and the protein regulation of nitrogen absorption and responses. NIN-like protein (NLP) transcription factors contain a Phox and Bem1 (PB1) domain, which may regulate nitrate-response genes and seem to be involved in the adaptation to growing on land in terms of plant root development. In this report, we reveal the NLP phylogeny in land plants and the origin of NLP genes that may be involved in the nitrate-signaling pathway. Our NLP phylogeny showed that duplication of NLP genes occurred before divergence of chlorophyte and land plants. Duplicated NLP genes may lost in most chlorophyte lineages. The NLP genes of bryophytes were initially monophyletic, but this was followed by divergence of lycophyte NLP genes and then angiosperm NLP genes. Among those identified NLP genes, PB1, a protein-protein interaction domain was identified across our phylogeny. To understand how protein-protein interaction mediate via PB1 domain, we examined the PB1 domain of Arabidopsis thaliana NLP7 (AtNLP7) in terms of its molecular oligomerization and function as representative. Based on the structure of the PB1 domain, determined using small-angle x-ray scattering (SAXS) and site-directed mutagenesis, we found that the NLP7 PB1 protein forms oligomers and that several key residues (K867 and D909/D911/E913/D922 in the OPCA motif) play a pivotal role in the oligomerization of NLP7 proteins. The fact that these residues are all conserved across land plant lineages means that this oligomerization may have evolved after the common ancestor of extant land plants colonized the land. It would then have rapidly become established across land-plant lineages in order to mediate protein-protein interactions in the nitrate-signaling pathway.

Generation and Characterization of a Spike Glycoprotein Domain A-Specific Neutralizing Single-Chain Variable Fragment against Porcine Epidemic Diarrhea Virus.

The emergence of the genotype (G) 2 and re-emergence of the G1 porcine epidemic diarrhea virus (PEDV) has caused severe economic impacts in the past decade. Developments of efficient vaccines against new variants of PEDV have been challenging, not least because of the difficulties in eliciting mucosal and lactogenic immunity. A single-chain fragment variable (scFv) capable of efficient antigen recognition is an alternative to vaccination and treatment of a viral infection. In the present study, the variable regions of the light chain and the heavy chain of a G2b PEDV spike domain A (S1A)-specific neutralizing monoclonal antibody (mAb) were sequenced, constructed with a (G4S) x3 linker, and produced by a mammalian protein expression system. Our results demonstrated that the PEDV S1A domain scFv was able to bind to S proteins of both G1 and G2b PEDVs. Nevertheless, the scFv was only capable of neutralizing the homologous G2b PEDV but not the G1 PEDV. The binding ability of the G2b-specific neutralizing scFv was not able to predict the neutralizing ability toward heterologous PEDV. The anti-PEDV S1A scFv presented herein serves as a potential therapeutic candidate against the virulent G2b PEDV.

Effect of SARS-CoV-2 B.1.1.7 mutations on spike protein structure and function.

The B.1.1.7 variant of SARS-CoV-2 first detected in the UK harbors amino-acid substitutions and deletions in the spike protein that potentially enhance host angiotensin conversion enzyme 2 (ACE2) receptor binding and viral immune evasion. Here we report cryo-EM structures of the spike protein of B.1.1.7 in the apo and ACE2-bound forms. The apo form showed one or two receptor-binding domains (RBDs) in the open conformation, without populating the fully closed state. All three RBDs were engaged in ACE2 binding. The B.1.1.7-specific A570D mutation introduces a molecular switch that could modulate the opening and closing of the RBD. The N501Y mutation introduces a π-π interaction that enhances RBD binding to ACE2 and abolishes binding of a potent neutralizing antibody (nAb). Cryo-EM also revealed how a cocktail of two nAbs simultaneously bind to all three RBDs, and demonstrated the potency of the nAb cocktail to neutralize different SARS-CoV-2 pseudovirus strains, including B.1.1.7.