Biology subjects

Walsh, R. M.

Publications and source records attributed to Walsh, R. M..

At least 19 recordsLinked to original sources

bioRxiv2026

Structure of Bacillus cereus ZmaM, a peptidase-ABC transporter that secretes and activates zwittermicin A

Bacteria engage in microbial warfare by secreting toxic molecules. These toxins can serve as therapeutics or drug discovery starting points, motivating investigations of their production. Bacteria that produce these toxins must protect themselves, and a common strategy is to synthesize an inactive prodrug that is activated upon secretion. Some prodrugs are capped with N-acyl-[x1D05]-asparagine motifs, which are removed by two types of prodrug peptidases. Type I peptidases are membrane-bound S12 hydrolases; type II peptidases have an additional C-terminal half-ABC transporter module. Recent structures of a type I peptidase revealed how its transmembrane domain completes the substrate-binding site, but little is known about type II peptidases. Here, we show that the B. cereus type II peptidase ZmaM is responsible for both the export of prezwittermicin and its activation into zwittermicin. The structure of homodimeric ZmaM reveals a central type IV ABC transporter flanked by peptidase modules. The periplasmic hydrolase domains interact over the transporter's open outer vestibule. Mutations to intermodule interfaces impact both peptidase and ATP hydrolysis activities, indicating the peptidase and transporter modules are interdependent. Together, our findings reveal an evolutionary strategy to coordinate toxin activation with its export and inform future efforts to harness these systems in biotechnology.

microbiology↗

bioRxiv2026

Timed binary WNT activity is sufficient to model the early human neural axis

The vertebrate nervous system develops into functionally distinct domains along the body axis[1]. Developmental studies implicate multiple signaling pathways in coordinating this process, but the order of specification events, sufficiency of individual signals, and molecular basis of competency remain unclear [2,3], leaving the field without a unified model of neuraxis patterning. Enteric nervous system (ENS) and midbrain dopaminergic (mDA) progenitors have reached clinical and preclinical testing [4-11], yet neither is produced at full purity, and yields vary markedly across human pluripotent stem cell (hPSC) lines, reflecting incomplete control of axial identity. Here we use single-cell genetic fate mapping to demonstrate that hPSCs can commit to anterior or posterior cranial fate within the first 24 hours of differentiation, before neural induction. Building on this finding, we show that the duration of a single saturating WNT pulse is sufficient to specify three 'first-order' domains: anterior cranial (0 h), posterior cranial (24 h) and trunk (48 h), each pre-neural and competent for CNS, neural crest and epidermal lineages. Nested 'second-order' domains are specified within first-order domains in response to WNT, FGF, and retinoic acid. Mechanistically, an anterior gene regulatory network repressing WNT targets is irreversibly shut down during the transition to posterior identities. Applying this binary WNT patterning logic, we generate mDA neurons and ENS progenitors robustly across hPSC lines and establish an assembloid model combining first-order progenitors to produce all major regions of the nervous system.

developmental biology↗

bioRxiv2026

Developmental rewiring of EHMT1 function during human cortical neurogenesis

Ubiquitously expressed chromatin regulators orchestrate cell-type-specific gene expression programs, yet how they acquire distinct regulatory functions during development remains fundamentally unresolved. Using temporally controlled protein depletion during human cortical neurogenesis in vitro, we show that the autism-associated histone methyltransferase EHMT1 is developmentally repurposed from epiblast to cortical neurons. Broadly expressed zinc finger proteins recruit EHMT1 to distinct cis-regulatory elements within changing chromatin landscapes associated with lineage-specific transcription factor activity, with extensive remodeling of EHMT1 enhancer occupancy during neural induction dependent on ZNF462. This genomic rewiring is accompanied by a fundamental shift from restraining premature developmental gene expression in pluripotent cells to supporting neuronal programs, with distinct transcriptional consequences of EHMT1 loss across neural progenitors and excitatory and inhibitory neuronal subtypes. Timed depletion and recovery experiments identify the neural progenitor stage as the onset of a developmental period during which EHMT1 is required for faithful neuronal maturation. Remarkably, EHMT1 can re-engage stage-specific targets after prolonged deficiency, allowing extensive transcriptional recovery with only limited molecular scarring. These findings reveal how ubiquitous epigenetic machinery acquires developmental specificity while demonstrating the resilience of developmental gene regulatory programs to prolonged disruption, with implications for neurodevelopmental disorders.

developmental biology↗

bioRxiv2026

Rapid and efficient oligodendrocyte differentiation from human pluripotent stem cells via dual inhibition of BMP and Notch signaling

The protracted timing required for oligodendrocyte differentiation from human pluripotent stem cells (hPSCs) has limited their use in disease modeling, drug screening, and cell therapy. In particular, the signals that drive oligodendrocyte specification and maturation after neural induction and ventral patterning remain poorly understood. Here, we present a protocol to derive human oligodendrocytes from hPSCs that is based solely on extrinsic cues, and we identify dual inhibition of BMP and Notch signaling as critical drivers of oligodendrocyte commitment and maturation. By day 42 of differentiation, up to 70% of the cells are positive for the oligodendrocyte marker O4, with minimal astrocyte contamination, and show robust expression of mature myelin markers including MBP, MOG, and MAG. These hPSC-derived oligodendrocytes closely match the molecular identity of primary fetal human oligodendrocytes as assessed by single-cell RNA sequencing and are functional as shown by in vitro myelination assays. In addition to the rapid generation of myelinating oligodendrocytes, the new protocol can be modularly adapted for the efficient production of PDGFR+ oligodendrocyte precursors or mixed glial populations containing AQP4+ astrocytes, thereby providing a cellular toolbox for the study of human glial lineages in translational applications.

developmental biology↗

bioRxiv2025

Effect of the S2' site cleavage on SARS-CoV-2 spike

SARS-CoV-2 initiates infection of host cells by fusing its envelope lipid bilayer with the cell membrane. To overcome kinetic barriers for membrane fusion, the virus-encoded spike (S) protein refolds from a metastable prefusion state to a lower energy, stable postfusion conformation. The protein is first split into S1 and S2 fragments at a proteolytic site after synthesis, and presumably further cleaved at a second site, known as the S2 site, before membrane fusion can occur. We report here a cryo-EM structure of S2 fragment after the S2 cleavage, possibly representing a late fusion intermediate conformation, in which the fusion peptide and transmembrane segment have yet to pack together, distinct from the final, postfusion state. Functional assays demonstrate that the S2 cleavage accelerates membrane fusion, probably by stabilizing fusion intermediates. These results advance our understanding of SARS-CoV-2 entry and may guide intervention strategies against pathogenetic coronaviruses.

biochemistry↗

bioRxiv2025

Non-Contrast microCT Analysis of Obese Adipose in Response to Cold-Exposure Reveals Sex-Specific Alterations

In 2020, it was reported that obesity in the United States had increased by 12% from 1999 to 2018. While exercise and diet are optimal lifestyle modifications to curb obesity, drug-based therapeutics focus on glucagon-like peptide (GLP) modifiers. Alternatively, current research suggests that a specialized type of adipose, called thermogenic adipose, may help protect against obesity. Active thermogenic adipose can metabolize free fatty acids (FFAs) and carbohydrates to carry out non-shivering thermogenesis (NST), potentially providing a method for reducing excess energy stores. While brown adipose tissue (BAT) provides the primary thermogenic response, we hypothesized that exposing diet-induced obese (DIO) mice to colder temperatures would also diminish white adipose tissue (WAT) depots and suppress their inflammatory signature. To measure adipose response to cold in vivo, we applied a non-contrast microCT ({micro}CT) imaging analysis. Male and female mice were housed at thermoneutrality (TN) and fed a Western-style diet (WD) ad lib until they became obese. Once they reached this stage, the mice were subjected to a thermalshift (TS) and exposed to either room temperature (RT) of 22{degrees}C or a colder temperature of 18{degrees}C. The adipose response was then assessed in post-exposure tissues by histological analysis, proteomics, and molecular characterization to correlate phenotypic changes with our {micro}CT findings. Results from this analysis revealed a sex-specific response to cold exposure: thermogenic adipose was predominantly formed in the interscapular BAT (iBAT) of male mice, while female mice showed formation in their perigonadal WAT (pgWAT) and iBAT when exposed to 18{degrees}C. Furthermore, male mice exhibited a decline in serum glucose levels when subjected to 18{degrees}C, which was increased in TS female mice. Serum-free fatty acids (FFAs) were unaffected by either sex across different environmental conditions. Importantly, using a mass-spectrometry-based approach, we detected a reduction in pro-inflammatory cytokines in the conditioned media (CM) of pgWAT and iBAT from TS male mice compared to TN DIO male mice. Overall, our studies demonstrated a new {micro}CT-based analytical method to detect changes in obese adipose tissue and highlighted unique sex-specific responses to environmental exposure. Our findings suggest that thermogenic adipose may offer a promising avenue for combating obesity and reducing its pathologic characteristics.

cell biology↗

bioRxiv2024

Cortical assembloids support the development of fast-spiking human PVALB+ cortical interneurons and uncover schizophrenia-associated defects

Disruption of parvalbumin positive (PVALB+) cortical interneurons is implicated in the pathogenesis of schizophrenia. However, how these defects emerge during brain development remains poorly understood. The protracted maturation of these cells during postnatal life has made their derivation from human pluripotent stem cells (hPSCs) extremely difficult, precluding hPSC-based disease modeling of their role in neuropsychiatric disease. Here we present a cortical assembloid system that supports the development of PVALB+ cortical interneurons which match the molecular profiles of primary PVALB+ interneurons and display their distinctive electrophysiological features. Further, we characterized cortical interneuron development in a series of CRISPR-generated isogenic structural variants associated with schizophrenia and identified variant-specific phenotypes affecting cortical interneuron migration and the molecular profile of PVALB+ cortical interneurons. These findings offer plausible mechanisms on how the disruption of cortical interneuron development may impact schizophrenia risk and provide the first human experimental platform to study of PVALB+ cortical interneurons.

neuroscience↗

bioRxiv2024

Octahedral small virus-like particles of dengue virus type 2

Flavivirus envelope (E) and precursor M (prM) proteins, when ectopically expressed, assemble into empty, virus-like particles (VLPs). Cleavage of prM to M and loss of the pr fragment converts the VLPs from immature to mature particles, mimicking a similar maturation of authentic virions. Most of the VLPs obtained by prM-E expression are smaller than virions; early, low-resolution cryo-EM studies suggested a simple, 60-subunit, icosahedral organization. We describe here the cryo-EM structure of immature, small VLPs from dengue virus type 2 and show that they have octahedral rather than icosahedral symmetry. The asymmetric unit of the octahedral particle is an asymmetric trimer of prM-E heterodimers, just as it is on icosahedral immature virions; the full, octahedrally symmetric particle thus has 24 such asymmetric trimers, or 72 prM-E heterodimers in all. Cleavage of prM and release of pr generates ovoid, somewhat irregular, mature particles. Previous work has shown that mature smVLPs have fusion properties identical to those of virions, consistent with local, virion-like clustering of 36 E dimers on their surface. The cryo-EM structure and the properties of these VLPs described here relate directly to on-going efforts to use them as vaccine immunogens. IMPORTANCEEctopic expression of flavivirus envelope (E) and precursor M (prM) proteins leads to formation and secretion of empty, virus-like particles (VLPs), which are candidate, non-infectious, virion-like components of flavivirus vaccines. We show that the immature particles of a major class of VLPs -- "small VLPs" (smVLPs), which have smaller diameter than those of virion size, -- are octahedrally (rather than icosahedrally) symmetric, with the same clustering of prM and E, as asymmetric trimers of prM-E heterodimers, found on immature virions. Cleavage of prM and formation of mature, smVLPs yields somewhat irregular, ovoid particles. Design and characterization of VLPs as vaccine components will need to take these properties into account.

microbiology↗

bioRxiv2024

Allosteric mechanism of membrane fusion activation in a herpesvirus

ABSTRACT/SUMMARYHerpesviridae infect nearly all humans for life, causing diseases that range from painful to life-threatening1. These viruses penetrate cells by employing a complex apparatus composed of separate receptor-binding, signal-transmitting, and membrane-fusing components2. But how these components coordinate their functions is unknown. Here, we determined the 4.19-angstrom cryoEM reconstruction of the central signal-transmitting component from herpes simplex virus 2, the gH/gL complex, in its elusive pre-activation state. Analysis of the continuum of conformational ensembles observed in cryoEM data revealed a series of structural rearrangements in gH/gL that allosterically transmit the fusion-triggering signal from the receptor-binding glycoprotein gD to the membrane fusogen gB. Furthermore, we identified a structural "switch" element in gH/gL that refolds and flips 180 degrees during the transition from pre-activation to activated form. Conservation of this "switch" in gH/gL homologs suggests that the proposed fusion triggering mechanism may apply to all Herpesviridae and points to a new target for subunit-based vaccines and treatment efforts.

microbiology↗

bioRxiv2024

Visualizing chaperone-mediated multistep assembly of the human 20S proteasome

Dedicated assembly factors orchestrate stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here, we report cryo-EM reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, and how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates, and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. The structural findings reported here explain many previous biochemical and genetic observations. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors, and reveals conceptual principles underlying human proteasome biogenesis.

biochemistry↗

bioRxiv2023

Structure of an insect gustatory receptor

Gustatory Receptors (GRs) are critical for insect chemosensation and are potential targets for controlling pests and disease vectors. However, GR structures have not been experimentally determined. We present structures of Bombyx mori Gr9 (BmGr9), a fructose-gated cation channel, in agonist-free and fructose-bound states. BmGr9 forms a tetramer similar to distantly related insect Olfactory Receptors (ORs). Upon fructose binding, BmGr9s ion channel gate opens through helix S7b movements. In contrast to ORs, BmGR9s ligand-binding pocket, shaped by a kinked helix S4 and a shorter extracellular S3-S4 loop, is larger and solvent accessible in both agonist-free and fructose-bound states. Also unlike ORs, fructose binding by BmGr9 involves helix S5 and a binding pocket lined with aromatic and polar residues. Structure-based sequence alignments reveal distinct patterns of ligand-binding pocket residue conservation in GR subfamilies associated with distinct ligand classes. These data provide insight into the molecular basis of GR ligand specificity and function.

biochemistry↗

bioRxiv2023

Combined PI3K and MAPK inhibition synergizes to suppress PDAC

Oncogenic KRAS mutations are nearly ubiquitous in pancreatic ductal adenocarcinoma (PDAC), yet therapeutic attempts to target KRAS as well as its target MAPK pathway effectors have shown limited success due to the difficulty to pharmacologically target KRAS, inherent drug resistance in PDAC cells, and acquired resistance through activation of alternative mitogenic pathways such JAK-STAT and PI3K-AKT. While KRAS canonically drives the MAPK signaling pathway via RAF-MEK-ERK, it is also known to play a role in PI3K-AKT signaling. Our therapeutic study targeted the PI3K-AKT pathway with the drug Omipalisib (p110/{beta}/{delta}/{gamma} and mTORC1/2 inhibitor) in combination with MAPK pathway targeting drug Trametinib (MEK1/2 inhibitor) or SHP099-HCL (SHP099), which is an inhibitor of the KRAS effector SHP2. Western blot analysis demonstrated that application of Trametinib or SHP099 alone selectively blocked ERK phosphorylation (pERK) but failed to suppress phosphorylated AKT (pAKT) and in some instances increased pAKT levels. Conversely, Omipalisib alone successfully inhibited pAKT but failed to suppress pERK. Therefore, we hypothesized that a combination therapeutic comprised of Omipalisib with either Trametinib or SHP099 would inhibit two prominent mitogenic pathways, MEK and PI3K-AKT, to more effectively suppress pancreatic cancer. In vitro studies demonstrated that both Omipalisib/Trametinib and Omipalisib/SHP099 combination therapeutic strategies were generally more effective than treatment with each drug individually at reducing proliferation, colony formation, and cell migration compared to vehicle controls. Additionally, we found that while combination Omipalisib/SHP099 treatment reduced implanted tumor growth in vivo, the Omipalisib/Trametinib treatment was significantly more effective. Therefore, we additionally tested the Omipalisib/Trametinib combination therapeutic in the highly aggressive PKT (Ptf1acre, LSL-KrasG12D, TGFbR2fl/fl) spontaneous mouse model of PDAC. We subsequently found that PKT mice treated with the Omipalisib/Trametinib combination therapeutic survived significantly longer than mice treated with either drug alone, and more than doubled the mean survival time of vehicle control mice. Altogether, our data support the importance of a dual treatment strategy targeting both MAPK and PI3K-AKT pathways.

cancer biology↗

bioRxiv2023

Adipose contributes to CXCL5 regulated immune evasion in pancreatic cancer

BackgroundCXCR1/2 inhibitors are being implemented with immunotherapies in PDAC clinical trials. Cytokines responsible for stimulating these receptors include CXCL ligands, typically secreted by activated immune cells, fibroblasts, and even adipocytes. Obesity has been linked to poor patient outcome and altered anti-tumor immunity. Adipose-derived cytokines and chemokines have been implicated as potential drivers of tumor cell immune evasion, suggesting a possibility of susceptibility to targeting specifically in the context of obesity. MethodsRNA-sequencing of human PDAC cell lines was used to assess differential influences on the cancer cell transcriptome after treatment with conditioned media from peri-pancreatic adipose tissue of lean and obese PDAC patients. The adipose-induced secretome of PDAC cells was then assessed by cytokine arrays and ELISAs. Lentiviral transduction and CRISPR-Cas9 was used to knock out CXCL5 from a murine PDAC cell line for orthotopic tumor studies in diet-induced obese, syngeneic mice. Flow cytometry was used to define the immune profiles of tumors. Anti-PD-1 immune checkpoint blockade therapy was administered to alleviate T cell exhaustion and invoke an immune response, while the mice were monitored at endpoint for differences in tumor size. ResultsThe chemokine CXCL5 was secreted in response to stimulation of PDAC cells with human adipose conditioned media (hAT-CM). PDAC CXCL5 secretion was induced by either IL-1{beta} or TNF, but neutralization of both was required to limit secretion. Ablation of CXCL5 from tumors promoted an immune phenotype susceptible to PD-1 inhibitor therapy. While application of anti-PD-1 treatment to control tumors failed to alter tumor growth, knockout CXCL5 tumors were diminished. ConclusionsIn summary, our findings show that known adipokines TNF and IL-1{beta} can stimulate CXCL5 release from PDAC cells in vitro. In vivo, CXCL5 depletion alone is sufficient to promote T cell infiltration into tumors in an obese setting, but requires checkpoint blockade inhibition to alleviate tumor burden. DATA AVAILABILITY STATEMENTRaw and processed RNAseq data will be further described in the GEO accession database (awaiting approval from GEO for PRJ number). Additional raw data is included in the supplemental material and available upon reasonable request. WHAT IS ALREADY KNOWN ON THIS TOPICObesity is linked to a worsened patient outcome and immunogenic tumor profile in PDAC. CXCR1/2 inhibitors have begun to be implemented in combination with immune checkpoint blockade therapies to promote T cell infiltration under the premise of targeting the myeloid rich TME. WHAT THIS STUDY ADDSUsing in vitro/ex vivo cell and tissue culture-based assays with in vivo mouse models we have identified that adipose derived IL-1{beta} and TNF can promote tumor secretion of CXCL5 which acts as a critical deterrent to CD8 T cell tumor infiltration, but loss of CXCL5 also leads to a more immune suppressive myeloid profile. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE, OR POLICYThis study highlights a mechanism and emphasizes the efficacy of single CXCR1/2 ligand targeting that could be beneficial to overcoming tumor immune-evasion even in the obese PDAC patient population.

cancer biology↗

bioRxiv2022

An epigenetic barrier sets the timing of human neuronal maturation

The pace of human brain development is highly protracted compared to most other species. The maturation of cortical neurons is particularly slow, extending over months to years to reach adult-like functions. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cell (hPSC) during in vitro differentiation or upon transplantation into the murine brain in vivo. Those findings suggest the presence of a cell intrinsic clock that sets the pace of neuronal maturation, though the molecular nature of such a clock has remained elusive. Here, we identify an epigenetic developmental program which sets the timing of human neuronal maturation. First, we developed a human PSC-based approach to synchronize the birth of cortical neurons in vitro which allowed us to define a detailed atlas of progressive morphological, functional, and molecular maturation in human cortical neurons. Interestingly, we observed a slow, temporal unfolding of maturation programs that is limited by the retention of a specific set of epigenetic factors. Loss-of-function studies for several of those factors in cortical neurons enables precocious molecular and physiological maturation. Remarkably, transient inhibition of EZH2, EHMT1/2 or DOT1L, at the progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Therefore, our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an "epigenetic barrier" in progenitor cells. Mechanistically, this barrier acts by holding transcriptional maturation programs in a poised state that gets gradually released during neuronal differentiation to ensure the prolonged timeline characteristic of human cortical neuron maturation.

developmental biology↗

bioRxiv2022

Combined small molecule treatment accelerates timing of maturation in human pluripotent stem cell-derived neurons

The maturation of human pluripotent stem cell (hPSC)-derived neurons mimics the protracted timing of human brain development, extending over months and years to reach adult-like function. Prolonged in vitro maturation presents a major challenge to stem cell-based applications in modeling and treating neurological disease. We designed a high-content imaging assay based on morphological and functional readouts in hPSC-derived cortical neurons to reveal underlying pathways and to identify chemicals capable of accelerating neuronal maturation. Probing a library of 2688 bioactive drugs, we identified multiple compounds that drive neuronal maturation including inhibitors of LSD1 and DOT1L and activators of calcium-dependent transcription. A cocktail of 4 factors GSK-2879552, EPZ-5676, NMDA and Bay K 8644, which we collectively termed GENtoniK, triggered maturation across all assays tested including measures of synaptic density, electrophysiology and transcriptomics. Remarkably, GENtoniK was similarly effective in enhancing neuronal maturation in 3D cortical organoids and in spinal motoneurons, and improved aspects of cell maturation in non-neural lineages such as melanocytes and pancreatic beta cells. These results demonstrate that the maturation of multiple hPSC-derived cell types can be enhanced by simple pharmacological intervention and suggests that some of the mechanisms controlling the timing of human maturation are shared across lineages.

neuroscience↗

bioRxiv2021

Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant

The Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has outcompeted previously prevalent variants and become a dominant strain worldwide. We report here structure, function and antigenicity of its full-length spike (S) trimer in comparison with those of other variants, including Gamma, Kappa, and previously characterized Alpha and Beta. Delta S can fuse membranes more efficiently at low levels of cellular receptor ACE2 and its pseudotyped viruses infect target cells substantially faster than all other variants tested, possibly accounting for its heightened transmissibility. Mutations of each variant rearrange the antigenic surface of the N-terminal domain of the S protein in a unique way, but only cause local changes in the receptor-binding domain, consistent with greater resistance particular to neutralizing antibodies. These results advance our molecular understanding of distinct properties of these viruses and may guide intervention strategies.

biochemistry↗

bioRxiv2021

Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants

Several fast-spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have become the dominant circulating strains that continue to fuel the COVID-19 pandemic despite intensive vaccination efforts throughout the world. We report here cryo-EM structures of the full-length spike (S) trimers of the B.1.1.7 and B.1.351 variants, as well as their biochemical and antigenic properties. Mutations in the B.1.1.7 protein increase the accessibility of its receptor binding domain and also the binding affinity for receptor angiotensin-converting enzyme 2 (ACE2). The enhanced receptor engagement can account for the increased transmissibility and risk of mortality as the variant may begin to infect efficiently infect additional cell types expressing low levels of ACE2. The B.1.351 variant has evolved to reshape antigenic surfaces of the major neutralizing sites on the S protein, rendering complete resistance to some potent neutralizing antibodies. These findings provide structural details on how the wide spread of SARS-CoV-2 enables rapid evolution to enhance viral fitness and immune evasion. They may guide intervention strategies to control the pandemic.

biochemistry↗

bioRxiv2020

Structural impact on SARS-CoV-2 spike protein by D614G substitution

Substitution for aspartic acid by glycine at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing pandemic, appears to facilitate rapid viral spread. The G614 variant has now replaced the D614-carrying virus as the dominant circulating strain. We report here cryo-EM structures of a full-length S trimer carrying G614, which adopts three distinct prefusion conformations differing primarily by the position of one receptor-binding domain (RBD). A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer, effectively increasing the number of functional spikes and enhancing infectivity. The loop transition may also modulate structural rearrangements of S protein required for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.

biochemistry↗