Biology subjects

Diskin, R.

Publications and source records attributed to Diskin, R..

13 recordsLinked to original sources

bioRxiv2026

Identification of a pan-orthoebolavirus-reactive antibody from an rVSV-EBOV vaccinated individual

Orthoebolaviruses such as Ebola virus (EBOV), Sudan virus (SUDV) and Bundibugyo virus (BDBV) can cause severe disease with high case-fatality rates. While licensed EBOV vaccines and therapeutic antibodies protect against EBOV infection, no single monoclonal antibody currently provides broad protection across multiple orthoebolaviruses. Here, we analyzed the humoral immune response of an rVSV-EBOV vaccinee to identify pan-orthoebolavirus-neutralizing antibodies. Using BDBV- and SUDV-glycoproteins for single B cell-sorting, we identified B10, which neutralized authentic EBOV and SUDV, with potent activity against SUDV compared with established cross-reactive antibodies. Structural analysis mapped antibody B10 binding to the pan-orthoebolavirus conserved GP2-stalk/HR2 region, associated with asymmetric trimer destabilization and spike opening. In vivo, B10 showed significant prophylactic efficacy in an EBOV mouse model and partial protection with antiviral activity in a SUDV mouse model. Together, these findings demonstrate that rVSV-EBOV vaccination induced the development of a broadly orthoebolavirus-neutralizing antibody that holds exeptional therapeutic potential.

immunology↗

bioRxiv2025

Structure-based design of antibody repertoires with drug-like properties

Animal immunization is the prevalent strategy for discovering antibody therapeutics1, but it is a lengthy and poorly controlled process. As an alternative, synthetic antibody repertoires deliver antibodies without animal welfare concerns, but the resulting antibodies often fail to exhibit "drug-like" biophysical properties1,2. Modern repertoires have improved developability by using a handful of frameworks with desirable biophysical properties, but at the cost of reduced structural diversity3-6. We developed a principled structure- and energy-based strategy, called CADAbRe, to navigate the complex tradeoffs between developability and structural diversity in repertoire design. The designed repertoire comprises billions of antibodies that are predicted to be stable and foldable, built from hundreds of different frameworks and hundreds of thousands of designed CDR H3s. We also developed an economical and scalable strategy for synthesizing large antibody repertoires, and as a proof of concept, designed and synthesized a 500-million variant phage display repertoire. Selections against four unrelated targets produced structurally diverse binders that exhibited drug-like properties, two of which were readily formatted as bispecifics for functional studies. Furthermore, one of the binders targets a challenging, highly charged surface. The proof-of-concept repertoire is available for academic research. We envision that the CADAbRe approach and repertoire will accelerate and rationalize antibody discovery while addressing animal-welfare concerns7.

synthetic biology↗

bioRxiv2025

The prefusion structure of the HERV-K (HML-2) Env spike complex

The human endogenous retrovirus K (HERV-K) is a retrovirus that got assimilated into the human genome in ancient times and has been inherited in our germline ever since. It enters cells using a class-I spike protein (Env) that mediates receptor recognition and membrane fusion. On top of having a biological role during development, HERV-K is activated in amyotrophic lateral sclerosis, various cancers, and other pathological conditions. Antibodies that target the HERV-K spike complex have therapeutic value, flagging the spike as a novel drug target. Here, we use cryo-EM to determine the trimeric structure of the HERV-K spike. The spike presents a distinct structure, which substantially differs from other class-I fusogens. Nevertheless, some general architectural features suggest a common origin with other retroviruses. Our structural analysis points to the putative receptor binding sites of the spike and provides insights into its function. The ability to structurally characterize the HERV-K spike may facilitate the development of antibody-based therapies. SignificanceRetroviruses integrate their genomes into host cell DNA. When this occurs in germline cells, the retroviral elements can be inherited and become part of the offspring genome. HERV-K is one such retrovirus that integrated into the human genome in ancient times. Over time, it gained a role in embryonic development but is also linked to various diseases, making it a potential drug target. To enter cells, HERV-K uses a spike protein, whose atomic structure we determined using electron microscopy. This structure reveals insights into the spikes function and HERV-Ks evolutionary ties with contemporary viruses. This structural information further provides a foundation for future drug development.

microbiology↗

bioRxiv2024

Potassium induces conformational changes in the Sabia virus spike complex

Hemorrhagic fever viruses from the Arenaviridae are a source of concern due to their potential to cause lethal outbreaks and the lack of effective therapeutics. Several Clade-B viruses, which are endemic to the Americas, are pathogens that sporadically infect humans following zoonotic transmission from small rodents. Brazilian hemorrhagic fever, caused by the Sabia virus (SBAV), is one of several such diseases from Clade-B arenaviruses. Despite their importance and the risks they impose, many fundamental questions remain regarding their biology and function. Here, we present the structure of the spike complex from the Sabia virus, which mediates viral attachment and entry to the host cells. Our study reveals two distinct conformational states of the spike, representing its native closed state and an open state that it assumes during cell entry. We show that potassium, in combination with acidic pH, promotes the opening of the spike, which is required for achieving efficient cell entry. This structure further informs us about the architecture of Clade-B arenaviral spikes and how they vary from spikes of other members of the Arenaviridae.

microbiology↗

bioRxiv2024

The Structure of the Lujo Virus Spike Complex

Lujo virus is a human pathogen that emerged as the etiology agent of a deadly viral disease in Africa. While it is a member of the Arenaviridae, it is a distinct virus that does not classify with the classical Old World or New World groups of viruses in this family. It further utilizes neuropilin-2 (NRP2) as an entry receptor, a property that is not shared by other arenaviruses. So far, structural information is limited to the receptor binding domain of LUJV, and the overall organization of the trimeric complex, as well as the way NRP2 is recognized in the context of the complete viral spike, were unknown. Here, we present the cryo-EM structure of the complete, native, membrane-embedded spike complex of LUJV. We found that NRP2 is bound at the apex of the spike in a way that allows each trimer to engage with a single NRP2. Also, the complete receptor binding site is quaternary, depending on interactions contributed by neighboring protomers. Recognition of NRP2 involves an overlooked arginine-methionine interaction, which we have now characterized. This LUJVs spike structure, which is the second determined structure of a complete arenaviral spike, points to similarities and differences in the structures of these viral spikes, informing vaccine design and allowing us to be better prepared to combat future outbreaks of this virus.

microbiology↗

bioRxiv2023

Dissecting the impact of somatic hypermutation on SARS-CoV-2 neutralization and viral escape

Somatic hypermutation (SHM) drives affinity maturation and continues over months in SARS-CoV-2 neutralizing antibodies. Yet, several potent SARS-CoV-2 antibodies carry no or only few mutations, leaving the question of how ongoing SHM affects neutralization. Here, we reverted variable region mutations of 92 antibodies and tested their impact on SARS-CoV-2 binding and neutralization. Reverting higher numbers of mutations correlated with decreasing antibody functionality. However, some antibodies, including the public clonotype VH1-58, remained unaffected for Wu01 activity. Moreover, while mutations were dispensable for Wu01-induced VH1-58 antibodies to neutralize Alpha, Beta, and Delta variants, they were critical to neutralize Omicron BA.1/BA.2. Notably, we exploited this knowledge to convert the clinical antibody tixagevimab into a BA.1/BA.2-neutralizer. These findings substantially broaden our understanding of SHM as a mechanism that not only improves antibody responses during affinity maturation, but also counteracts antigenic imprinting through antibody diversification and thus increases the chances of neutralizing viral escape variants.

immunology↗

bioRxiv2022

The homodimeric structure of the LARGE1 dual glycosyltransferase

LARGE1 is a bifunctional glycosyltransferase responsible for generating a long linear polysaccharide termed matriglycan that links the cytoskeleton and the extracellular matrix and is required for proper muscle function. This matriglycan polymer is made with an alternating pattern of xylose and glucuronic acid monomers. Mutations in the LARGE1 gene have been shown to cause life-threatening dystroglycanopathies through the inhibition of matriglycan synthesis. Despite its major role in muscle maintenance, the structure of the LARGE1 enzyme and how it assembles in the Golgi are unknown. Here we present the structure of LARGE1, obtained by a combination of X-ray crystallography and single-particle cryo-EM. We found that LARGE1 homo-dimerizes in a configuration that is dictated by its coiled-coil stem domain. The structure shows that this enzyme has two canonical GT-A folds with each of its catalytic domains. In the context of its dimeric structure, the two types of catalytic domains are brought into close proximity from opposing monomers to allow efficient shuttling of the substrate between the two domains. Together with putative retention of matriglycan by electrostatic interactions, this dimeric organization offers a possible mechanism for the high processivity of LARGE1. The structural information further reveals the mechanisms in which disease-causing mutations disrupt the activity of LARGE1. Collectively, these data shed light on how matriglycan is synthesized alongside the functional significance of glycosyltransferase oligomerization.

molecular biology↗

bioRxiv2021

Biomolecular recognition of the glycan neoantigen CA19-9 by distinct antibodies

Glycans decorate cell surface, secreted glycoproteins and glycolipids. Altered glycans are often found in cancers. Despite their high diagnostic and therapeutic potentials, glycans are polar and flexible molecules that are quite challenging for the development and design of high-affinity binding antibodies. To understand the mechanisms by which glycan neoantigens are specifically recognized by antibodies, we analyze the biomolecular recognition of a single tumor-associated carbohydrate antigen CA19-9 by two distinct antibodies using X-ray crystallography. Despite the plasticity of glycans and the very different antigen-binding surfaces presented by the antibodies, both structures reveal an essentially identical extended CA19-9 conformer, suggesting that this conformers stability selects the antibodies. Starting from the bound structure of one of the antibodies, we use the AbLIFT computational method to design a variant with seven core mutations that exhibited tenfold improved affinity for CA19-9. The results reveal strategies used by antibodies to specifically recognize glycan antigens and show how automated antibody-optimization methods may be used to enhance the clinical potential of existing antibodies.

molecular biology↗

bioRxiv2020

Coronacept - a potent immunoadhesin against SARS-CoV-2

Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Computational analysis of mammalian ACE2 orthologues suggests various residues at the interface with the viral receptor binding domain that could facilitate tighter interaction compared to the human-ACE2. Introducing several mutations to the human-ACE2 resulted with significantly augmented affinity to the viral spike complex. This modified human-ACE2 fused to an Fc portion of an antibody makes a potent immunoadhesin that effectively targets SARS-CoV-2.

bioengineering↗

bioRxiv2020

Longitudinal isolation of potent near-germline SARS-CoV-2-neutralizing antibodies from COVID-19 patients

The SARS-CoV-2 pandemic has unprecedented implications for public health, social life, and world economy. Since approved drugs and vaccines are not available, new options for COVID-19 treatment and prevention are highly demanded. To identify SARS-CoV-2 neutralizing antibodies, we analysed the antibody response of 12 COVID-19 patients from 8 to 69 days post diagnosis. By screening 4,313 SARS-CoV-2-reactive B cells, we isolated 255 antibodies from different time points as early as 8 days post diagnosis. Among these, 28 potently neutralized authentic SARS-CoV-2 (IC100 as low as 0.04 g/ml), showing a broad spectrum of V genes and low levels of somatic mutations. Interestingly, potential precursors were identified in naive B cell repertoires from 48 healthy individuals that were sampled before the COVID-19 pandemic. Our results demonstrate that SARS-CoV-2 neutralizing antibodies are readily generated from a diverse pool of precursors, fostering the hope of rapid induction of a protective immune response upon vaccination.

immunology↗

bioRxiv2020

Assembly Mechanism of Mucin and von Willebrand Factor Polymers

The respiratory and intestinal tracts are exposed to physical and biological hazards accompanying the intake of air and food. Likewise, the vasculature is threatened by inflammation and trauma. Mucin glycoproteins and the related von Willebrand factor (VWF) guard the vulnerable cell layers in these diverse systems. Colon mucins additionally house and feed the gut microbiome. Here we present an integrated structural analysis of multimerized intestinal mucin MUC2. Our findings reveal the shared mechanism by which complex macromolecules responsible for blood clotting, mucociliary clearance, and the intestinal mucosal barrier form protective polymers and hydrogels. Specifically, cryo-electron microscopy and crystal structures show how disulfide-rich bridges and pH-tunable interfaces control successive assembly steps in the endoplasmic reticulum and Golgi. Remarkably, a densely O-glycosylated mucin domain performs a specific organizational role in MUC2. The mucin assembly mechanism and its adaptation for hemostasis provide the foundation for rational manipulation of barrier function and coagulation.

molecular biology↗

bioRxiv2018

Expression of a recombinant, 4’-Phosphopantetheinylated, active M. tuberculosis Fatty acid Synthase I in E. coli

Fatty acid synthase 1 (FAS I) from Mycobacterium. tuberculosis (Mtb) is an essential protein and a promising drug target. FAS I is a multi-functional, multi-domain protein that is organized as a large (1.9 MDa) homohexameric complex. Acyl intermediates produced during fatty acid elongation are attached covalently to an acyl carrier protein (ACP) domain. This domain is activated by the transfer of a 4-Phosphopantetheine (4-PP, also termed P-pant) group from CoA to ACP catalyzed by a 4-PP transferase, termed acyl carrier protein synthase (AcpS). In order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fas1 and acpS genes encoded by a separate plasmid.\n\nWe induced the expression of Mtb FAS I following induction of AcpS expression. FAS I was purified by Strep-Tactin affinity chromatography. Activation of Mtb FAS I was confirmed by the identification of a bound P-pant group on serine at position 1808 by mass spectrometry. The purified FAS I displayed biochemical activity shown by spectrophotometric analysis of NADPH oxidation and by CoA production, using the Ellman reaction. The purified Mtb FAS I forms a hexameric complex shown by negative staining and cryo-EM. Purified hexameric and active Mtb FAS I is required for binding and drug inhibition studies and for structurefunction analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.

microbiology↗

bioRxiv2018

Structural Basis for Receptor Recognition by Lujo Virus

Lujo virus (LUJV) has emerged as a novel and highly fatal human pathogen. Despite its membership among the Arenaviridae, LUJV does not classify with the known Old and New World groups of that viral family. Likewise, LUJV was recently found to use neuropilin-2 (NRP2) as a cellular receptor instead of the canonical -dystroglycan (-DG) or transferrin receptor 1 (TfR1) utilized by Old World (OW) and New World (NW) arenaviruses, respectively. The emergence of a deadly new pathogen into human populations using an unprecedented entry route raises many questions regarding the mechanism of cell recognition and the risk that Arenaviruses are further diversifying their infection strategies. To provide the basis for combating LUJV in particular, and to increase our general understanding of the molecular changes that accompany an evolutionary switch to a new receptor for Arenaviruses, we used X-ray crystallography to reveal how the GP1 receptor-binding domain of LUJV (LUJVGP1) recognizes NRP2. Our structural data imply that LUJV is evolutionary closer to OW than to NW arenaviruses. Structural analysis supported by experimental validation further suggests that NRP2 recognition is metal ion dependent and that the complete NRP2 binding is formed in the context of the trimeric spike. Taken together, our data provide the mechanism for the cell attachment step of LUJV, the evolutionary relationship between the GP1 domain of this novel pathogen and other arenaviruses, and indispensable information for combating LUJV.

microbiology↗