35 results on '"Binshtein E"'
Search Results
2. CryoEM structure of Eastern Equine Encephalitis (EEEV) VLP
- Author
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Binshtein, E., primary and Crowe, J.E., additional
- Published
- 2020
- Full Text
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3. CryoEM structure of Eastern Equine Encephalitis (EEEV) VLP with Fab EEEV-143.
- Author
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Binshtein, E., primary and Crowe, J.E., additional
- Published
- 2020
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4. Can glyoxylate carboligase be a promiscuous enzyme?: YSF-9
- Author
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Binshtein, E., Temam, V., Shaanan, B., Chipman, D. M., and Barak, Z.
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- 2009
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5. Functional and structural basis of human parainfluenza virus type 3 neutralization with human monoclonal antibodies.
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Suryadevara N, Otrelo-Cardoso AR, Kose N, Hu YX, Binshtein E, Wolters RM, Greninger AL, Handal LS, Carnahan RH, Moscona A, Jardetzky TS, and Crowe JE Jr
- Subjects
- Animals, Humans, Disease Models, Animal, Neutralization Tests, B-Lymphocytes immunology, Models, Molecular, Parainfluenza Virus 3, Human immunology, Parainfluenza Virus 3, Human genetics, Antibodies, Monoclonal immunology, Antibodies, Monoclonal chemistry, Antibodies, Neutralizing immunology, Antibodies, Neutralizing chemistry, Antibodies, Viral immunology, Antibodies, Viral chemistry, Sigmodontinae, Viral Fusion Proteins immunology, Viral Fusion Proteins chemistry, HN Protein immunology, HN Protein chemistry, HN Protein genetics, Respirovirus Infections immunology, Respirovirus Infections virology
- Abstract
Human parainfluenza virus type 3 (hPIV3) is a respiratory pathogen that can cause severe disease in older people and infants. Currently, vaccines against hPIV3 are in clinical trials but none have been approved yet. The haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins of hPIV3 are major antigenic determinants. Here we describe naturally occurring potently neutralizing human antibodies directed against both surface glycoproteins of hPIV3. We isolated seven neutralizing HN-reactive antibodies and a pre-fusion conformation F-reactive antibody from human memory B cells. One HN-binding monoclonal antibody (mAb), designated PIV3-23, exhibited functional attributes including haemagglutination and neuraminidase inhibition. We also delineated the structural basis of neutralization for two HN and one F mAbs. MAbs that neutralized hPIV3 in vitro protected against infection and disease in vivo in a cotton rat model of hPIV3 infection, suggesting correlates of protection for hPIV3 and the potential clinical utility of these mAbs., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)
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- 2024
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6. Computationally restoring the potency of a clinical antibody against Omicron.
- Author
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Desautels TA, Arrildt KT, Zemla AT, Lau EY, Zhu F, Ricci D, Cronin S, Zost SJ, Binshtein E, Scheaffer SM, Dadonaite B, Petersen BK, Engdahl TB, Chen E, Handal LS, Hall L, Goforth JW, Vashchenko D, Nguyen S, Weilhammer DR, Lo JK, Rubinfeld B, Saada EA, Weisenberger T, Lee TH, Whitener B, Case JB, Ladd A, Silva MS, Haluska RM, Grzesiak EA, Earnhart CG, Hopkins S, Bates TW, Thackray LB, Segelke BW, Lillo AM, Sundaram S, Bloom JD, Diamond MS, Crowe JE Jr, Carnahan RH, and Faissol DM
- Subjects
- Animals, Female, Humans, Mice, COVID-19 immunology, COVID-19 virology, Mutation, Neutralization Tests, Spike Glycoprotein, Coronavirus chemistry, Spike Glycoprotein, Coronavirus genetics, Spike Glycoprotein, Coronavirus immunology, DNA Mutational Analysis, Antigenic Drift and Shift genetics, Antigenic Drift and Shift immunology, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal immunology, Antibodies, Neutralizing chemistry, Antibodies, Neutralizing immunology, Antibodies, Viral chemistry, Antibodies, Viral immunology, SARS-CoV-2 classification, SARS-CoV-2 genetics, SARS-CoV-2 immunology, Drug Design methods, Computer Simulation
- Abstract
The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs
1-3 and revealed how quickly viral escape can curtail effective options4,5 . When the SARS-CoV-2 Omicron variant emerged in 2021, many antibody drug products lost potency, including Evusheld and its constituent, cilgavimab4-6 . Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign and renew the efficacy of COV2-2130 against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and subsequent variants of concern, and provides protection in vivo against the strains tested: WA1/2020, BA.1.1 and BA.5. Deep mutational scanning of tens of thousands of pseudovirus variants reveals that 2130-1-0114-112 improves broad potency without increasing escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Our computational approach does not require experimental iterations or pre-existing binding data, thus enabling rapid response strategies to address escape variants or lessen escape vulnerabilities., (© 2024. The Author(s).)- Published
- 2024
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7. SARS-CoV-2 antibodies from children exhibit broad neutralization and belong to adult public clonotypes.
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Wall SC, Suryadevara N, Kim C, Shiakolas AR, Holt CM, Irbe EB, Wasdin PT, Suresh YP, Binshtein E, Chen EC, Zost SJ, Canfield E, Crowe JE Jr, Thompson-Arildsen MA, Sheward DJ, Carnahan RH, and Georgiev IS
- Subjects
- Humans, Adult, Child, SARS-CoV-2 genetics, Antibodies, Viral, Antibodies, Neutralizing therapeutic use, Pandemics, COVID-19
- Abstract
From the beginning of the COVID-19 pandemic, children have exhibited different susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, reinfection, and disease compared with adults. Motivated by the established significance of SARS-CoV-2-neutralizing antibodies in adults, here we characterize SARS-CoV-2-specific antibody repertoires in a young cohort of individuals aged from 5 months to 18 years old. Our results show that neutralizing antibodies in children possess similar genetic features compared to antibodies identified in adults, with multiple antibodies from children belonging to previously established public antibody clonotypes in adults. Notably, antibodies from children show potent neutralization of circulating SARS-CoV-2 variants that have cumulatively resulted in resistance to virtually all approved monoclonal antibody therapeutics. Our results show that children can rely on similar SARS-CoV-2 antibody neutralization mechanisms compared to adults and are an underutilized source for the discovery of effective antibody therapeutics to counteract the ever-evolving pandemic., Competing Interests: Declaration of interests S.C.W. and I.S.G. are listed as inventors on antibodies described here. A.R.S. and I.S.G. are co-founders of AbSeek Bio. I.S.G. and A.R.S. are listed as inventors on patent applications for the LIBRA-seq technology. D.J.S. has served as a consultant for AstraZeneca AB. J.E.C. has served as a consultant for Luna Biologics, is a member of the Scientific Advisory Board of Meissa Vaccines, and is founder of IDBiologics. The Crowe laboratory has received funding support in sponsored research agreements from AstraZeneca, IDBiologics, and Takeda. The Georgiev laboratory at VUMC has received unrelated funding from Takeda Pharmaceuticals., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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8. Multifunctional human monoclonal antibody combination mediates protection against Rift Valley fever virus at low doses.
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Chapman NS, Hulswit RJG, Westover JLB, Stass R, Paesen GC, Binshtein E, Reidy JX, Engdahl TB, Handal LS, Flores A, Gowen BB, Bowden TA, and Crowe JE Jr
- Subjects
- Animals, Mice, Humans, Biological Assay, Disease Models, Animal, Low Density Lipoprotein Receptor-Related Protein-1, Antibodies, Monoclonal, Rift Valley fever virus
- Abstract
The zoonotic Rift Valley fever virus (RVFV) can cause severe disease in humans and has pandemic potential, yet no approved vaccine or therapy exists. Here we describe a dual-mechanism human monoclonal antibody (mAb) combination against RVFV that is effective at minimal doses in a lethal mouse model of infection. We structurally analyze and characterize the binding mode of a prototypical potent Gn domain-A-binding antibody that blocks attachment and of an antibody that inhibits infection by abrogating the fusion process as previously determined. Surprisingly, the Gn domain-A antibody does not directly block RVFV Gn interaction with the host receptor low density lipoprotein receptor-related protein 1 (LRP1) as determined by a competitive assay. This study identifies a rationally designed combination of human mAbs deserving of future investigation for use in humans against RVFV infection. Using a two-pronged mechanistic approach, we demonstrate the potent efficacy of a rationally designed combination mAb therapeutic., (© 2023. Springer Nature Limited.)
- Published
- 2023
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9. Systematic analysis of human antibody response to ebolavirus glycoprotein shows high prevalence of neutralizing public clonotypes.
- Author
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Chen EC, Gilchuk P, Zost SJ, Ilinykh PA, Binshtein E, Huang K, Myers L, Bonissone S, Day S, Kona CR, Trivette A, Reidy JX, Sutton RE, Gainza C, Diaz S, Williams JK, Selverian CN, Davidson E, Saphire EO, Doranz BJ, Castellana N, Bukreyev A, Carnahan RH, and Crowe JE Jr
- Subjects
- Humans, Animals, Mice, Antibodies, Neutralizing, Antibodies, Viral, Antibody Formation, Prevalence, Glycoproteins genetics, Ebolavirus, Hemorrhagic Fever, Ebola
- Abstract
Understanding the human antibody response to emerging viral pathogens is key to epidemic preparedness. As the size of the B cell response to a pathogenic-virus-protective antigen is poorly defined, we perform deep paired heavy- and light-chain sequencing in Ebola virus glycoprotein (EBOV-GP)-specific memory B cells, allowing analysis of the ebolavirus-specific antibody repertoire both genetically and functionally. This approach facilitates investigation of the molecular and genetic basis for the evolution of cross-reactive antibodies by elucidating germline-encoded properties of antibodies to EBOV and identification of the overlap between antibodies in the memory B cell and serum repertoire. We identify 73 public clonotypes of EBOV, 20% of which encode antibodies with neutralization activity and capacity to protect mice in vivo. This comprehensive analysis of the public and private antibody repertoire provides insight into the molecular basis of the humoral immune response to EBOV GP, which informs the design of vaccines and improved therapeutics., Competing Interests: Declaration of interests E.D. and B.J.D. are employees of Integral Molecular, and B.J.D.is a shareholder in that company. J.E.C. has served as a consultant for Luna Labs USA, Merck Sharp & Dohme, Emergent Biosolutions, and GlaxoSmithKline; is a member of the scientific advisory board of Meissa Vaccines; is a former member of the scientific advisory board of Gigagen (Grifols); and is the founder of IDBiologics. The laboratory of J.E.C. received unrelated sponsored research agreements from AstraZeneca, Takeda, and IDBiologics during the conduct of the study., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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10. Computationally restoring the potency of a clinical antibody against SARS-CoV-2 Omicron subvariants.
- Author
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Desautels TA, Arrildt KT, Zemla AT, Lau EY, Zhu F, Ricci D, Cronin S, Zost SJ, Binshtein E, Scheaffer SM, Dadonaite B, Petersen BK, Engdahl TB, Chen E, Handal LS, Hall L, Goforth JW, Vashchenko D, Nguyen S, Weilhammer DR, Lo JK, Rubinfeld B, Saada EA, Weisenberger T, Lee TH, Whitener B, Case JB, Ladd A, Silva MS, Haluska RM, Grzesiak EA, Earnhart CG, Hopkins S, Bates TW, Thackray LB, Segelke BW, Lillo AM, Sundaram S, Bloom J, Diamond MS, Crowe JE Jr, Carnahan RH, and Faissol DM
- Abstract
The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs
1-3 , but also revealed how quickly viral escape can curtail effective options4,5 . With the emergence of the SARS-CoV-2 Omicron variant in late 2021, many clinically used antibody drug products lost potency, including Evusheld™ and its constituent, cilgavimab4,6 . Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies with a known clinical profile to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign COV2-2130 to rescue in vivo efficacy against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the contemporaneously dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and many variants of concern that subsequently emerged, and provides protection in vivo against the strains tested, WA1/2020, BA.1.1, and BA.5. Deep mutational scanning of tens of thousands pseudovirus variants reveals 2130-1-0114-112 improves broad potency without incurring additional escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Because our approach is computationally driven, not requiring experimental iterations or pre-existing binding data, it could enable rapid response strategies to address escape variants or pre-emptively mitigate escape vulnerabilities., Competing Interests: COMPETING FINANCIAL INTERESTS M.S.D. is a consultant for Inbios, Vir Biotechnology, Ocugen, Moderna and Immunome. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. J.E.C. has served as a consultant for Luna Labs USA, Merck Sharp & Dohme Corporation, Emergent Biosolutions, and GlaxoSmithKline, is a member of the Scientific Advisory Board of Meissa Vaccines, a former member of the Scientific Advisory Board of Gigagen (Grifols) and is founder of IDBiologics. The laboratory of J.E.C. received unrelated sponsored research agreements from AstraZeneca, Takeda, and IDBiologics during the conduct of the study. J. D. B. is on the scientific advisory boards of Apriori Bio, Aerium Therapuetics, Invivyd, and the Vaccine Company. Lawrence Livermore National Laboratory, Los Alamos National Laboratory, and Vanderbilt University have applied for patents for some of the antibodies in this paper, for which T.A.D, K.T.A, A.T.Z., E.Y.L., F.Z., A.M.L., R.H.C., J.E.C., and D.M.F. are inventors. Vanderbilt University has licensed certain rights to antibodies in this paper to Astra Zeneca. J. D. B. and B.D. are inventors on Fred Hutch licensed patents related to the deep mutational scanning of viral proteins.- Published
- 2023
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11. Antigenic mapping and functional characterization of human New World hantavirus neutralizing antibodies.
- Author
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Engdahl TB, Binshtein E, Brocato RL, Kuzmina NA, Principe LM, Kwilas SA, Kim RK, Chapman NS, Porter MS, Guardado-Calvo P, Rey FA, Handal LS, Diaz SM, Zagol-Ikapitte IA, Tran MH, McDonald WH, Meiler J, Reidy JX, Trivette A, Bukreyev A, Hooper JW, and Crowe JE
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- Animals, Humans, Antibodies, Neutralizing, Antibodies, Viral, Rodentia, Orthohantavirus, Hantavirus Infections prevention & control, Hantaan virus, Communicable Diseases
- Abstract
Hantaviruses are high-priority emerging pathogens carried by rodents and transmitted to humans by aerosolized excreta or, in rare cases, person-to-person contact. While infections in humans are relatively rare, mortality rates range from 1 to 40% depending on the hantavirus species. There are currently no FDA-approved vaccines or therapeutics for hantaviruses, and the only treatment for infection is supportive care for respiratory or kidney failure. Additionally, the human humoral immune response to hantavirus infection is incompletely understood, especially the location of major antigenic sites on the viral glycoproteins and conserved neutralizing epitopes. Here, we report antigenic mapping and functional characterization for four neutralizing hantavirus antibodies. The broadly neutralizing antibody SNV-53 targets an interface between Gn/Gc, neutralizes through fusion inhibition and cross-protects against the Old World hantavirus species Hantaan virus when administered pre- or post-exposure. Another broad antibody, SNV-24, also neutralizes through fusion inhibition but targets domain I of Gc and demonstrates weak neutralizing activity to authentic hantaviruses. ANDV-specific, neutralizing antibodies (ANDV-5 and ANDV-34) neutralize through attachment blocking and protect against hantavirus cardiopulmonary syndrome (HCPS) in animals but target two different antigenic faces on the head domain of Gn. Determining the antigenic sites for neutralizing antibodies will contribute to further therapeutic development for hantavirus-related diseases and inform the design of new broadly protective hantavirus vaccines., Competing Interests: TE, EB, RB, NK, LP, SK, RK, NC, MP, PG, FR, LH, SD, IZ, MT, WM, JM, JR, AT, AB, JH No competing interests declared, JC has served as a consultant for Luna Labs USA, Merck Sharp & Dohme Corporation, Emergent Biosolutions, GlaxoSmithKline and BTG International Inc, is a member of the Scientific Advisory Board of Meissa Vaccines, a former member of the Scientific Advisory Board of Gigagen (Grifols) and is founder of IDBiologics. The laboratory of J.E.C. received unrelated sponsored research agreements from AstraZeneca, Takeda, and IDBiologics during the conduct of the study
- Published
- 2023
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12. Functional HIV-1/HCV cross-reactive antibodies isolated from a chronically co-infected donor.
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Pilewski KA, Wall S, Richardson SI, Manamela NP, Clark K, Hermanus T, Binshtein E, Venkat R, Sautto GA, Kramer KJ, Shiakolas AR, Setliff I, Salas J, Mapengo RE, Suryadevara N, Brannon JR, Beebout CJ, Parks R, Raju N, Frumento N, Walker LM, Fechter EF, Qin JS, Murji AA, Janowska K, Thakur B, Lindenberger J, May AJ, Huang X, Sammour S, Acharya P, Carnahan RH, Ross TM, Haynes BF, Hadjifrangiskou M, Crowe JE Jr, Bailey JR, Kalams S, Morris L, and Georgiev IS
- Subjects
- Humans, Hepacivirus, Antibodies, Neutralizing, SARS-CoV-2, HIV Antibodies, HIV-1, Coinfection, HIV Infections, COVID-19, Hepatitis C
- Abstract
Despite prolific efforts to characterize the antibody response to human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) mono-infections, the response to chronic co-infection with these two ever-evolving viruses is poorly understood. Here, we investigate the antibody repertoire of a chronically HIV-1/HCV co-infected individual using linking B cell receptor to antigen specificity through sequencing (LIBRA-seq). We identify five HIV-1/HCV cross-reactive antibodies demonstrating binding and functional cross-reactivity between HIV-1 and HCV envelope glycoproteins. All five antibodies show exceptional HCV neutralization breadth and effector functions against both HIV-1 and HCV. One antibody, mAb688, also cross-reacts with influenza and coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We examine the development of these antibodies using next-generation sequencing analysis and lineage tracing and find that somatic hypermutation established and enhanced this reactivity. These antibodies provide a potential future direction for therapeutic and vaccine development against current and emerging infectious diseases. More broadly, chronic co-infection represents a complex immunological challenge that can provide insights into the fundamental rules that underly antibody-antigen specificity., Competing Interests: Declaration of interests K.A.P. and I.S.G. are listed as inventors on patents filed describing the antibodies discovered here. A.R.S. and I.S.G. are co-founders of AbSeek Bio. J.E.C. has served as a consultant for Luna Biologics, is a member of the Scientific Advisory Board of Meissa Vaccines, and is founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from Takeda Vaccines, IDBiologics, and AstraZeneca. The Georgiev laboratory at Vanderbilt University Medical Center has received unrelated funding from Takeda Pharmaceuticals., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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13. An antibody targeting the N-terminal domain of SARS-CoV-2 disrupts the spike trimer.
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Suryadevara N, Shiakolas AR, VanBlargan LA, Binshtein E, Chen RE, Case JB, Kramer KJ, Armstrong EC, Myers L, Trivette A, Gainza C, Nargi RS, Selverian CN, Davidson E, Doranz BJ, Diaz SM, Handal LS, Carnahan RH, Diamond MS, Georgiev IS, and Crowe JE Jr
- Subjects
- Animals, Antibodies, Neutralizing, Antibodies, Viral, Humans, Mice, Spike Glycoprotein, Coronavirus genetics, COVID-19 genetics, SARS-CoV-2 genetics
- Abstract
The protective human antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) focuses on the spike (S) protein, which decorates the virion surface and mediates cell binding and entry. Most SARS-CoV-2 protective antibodies target the receptor-binding domain or a single dominant epitope ("supersite") on the N-terminal domain (NTD). Using the single B cell technology called linking B cell receptor to antigen specificity through sequencing (LIBRA-Seq), we isolated a large panel of NTD-reactive and SARS-CoV-2-neutralizing antibodies from an individual who had recovered from COVID-19. We found that neutralizing antibodies against the NTD supersite were commonly encoded by the IGHV1-24 gene, forming a genetic cluster representing a public B cell clonotype. However, we also discovered a rare human antibody, COV2-3434, that recognizes a site of vulnerability on the SARS-CoV-2 S protein in the trimer interface (TI) and possesses a distinct class of functional activity. COV2-3434 disrupted the integrity of S protein trimers, inhibited the cell-to-cell spread of the virus in culture, and conferred protection in human angiotensin-converting enzyme 2-transgenic (ACE2-transgenic) mice against the SARS-CoV-2 challenge. This study provides insight into antibody targeting of the S protein TI region, suggesting this region may be a site of virus vulnerability.
- Published
- 2022
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14. Epitope-focused immunogen design based on the ebolavirus glycoprotein HR2-MPER region.
- Author
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Schoeder CT, Gilchuk P, Sangha AK, Ledwitch KV, Malherbe DC, Zhang X, Binshtein E, Williamson LE, Martina CE, Dong J, Armstrong E, Sutton R, Nargi R, Rodriguez J, Kuzmina N, Fiala B, King NP, Bukreyev A, Crowe JE Jr, and Meiler J
- Subjects
- Animals, Antibodies, Neutralizing, Antibodies, Viral, Epitopes, Glycoproteins, Rabbits, Ebolavirus, Hemorrhagic Fever, Ebola
- Abstract
The three human pathogenic ebolaviruses: Zaire (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) virus, cause severe disease with high fatality rates. Epitopes of ebolavirus glycoprotein (GP) recognized by antibodies with binding breadth for all three ebolaviruses are of major interest for rational vaccine design. In particular, the heptad repeat 2 -membrane-proximal external region (HR2-MPER) epitope is relatively conserved between EBOV, BDBV, and SUDV GP and targeted by human broadly-neutralizing antibodies. To study whether this epitope can serve as an immunogen for the elicitation of broadly-reactive antibody responses, protein design in Rosetta was employed to transplant the HR2-MPER epitope identified from a co-crystal structure with the known broadly-reactive monoclonal antibody (mAb) BDBV223 onto smaller scaffold proteins. From computational analysis, selected immunogen designs were produced as recombinant proteins and functionally validated, leading to the identification of a sterile alpha motif (SAM) domain displaying the BDBV-HR2-MPER epitope near its C terminus as a promising candidate. The immunogen was fused to one component of a self-assembling, two-component nanoparticle and tested for immunogenicity in rabbits. Robust titers of cross-reactive serum antibodies to BDBV and EBOV GPs and moderate titers to SUDV GP were induced following immunization. To confirm the structural composition of the immunogens, solution NMR studies were conducted and revealed structural flexibility in the C-terminal residues of the epitope. Overall, our study represents the first report on an epitope-focused immunogen design based on the structurally challenging BDBV-HR2-MPER epitope., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: J.E.C. has served as a consultant for Takeda Vaccines, Sanofi-Aventis U.S., Pfizer, Novavax, Lilly and Luna Biologics, is a member of the Scientific Advisory Boards of CompuVax and Meissa Vaccines and is Founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from IDBiologics. N.P.K. is a co-founder, shareholder, paid consultant, and chair of the scientific advisory board of Icosavax, Inc. and has received an unrelated sponsored research agreement from Pfizer. All other authors declare no conflict of interest.
- Published
- 2022
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15. Neutralizing antibodies protect mice against Venezuelan equine encephalitis virus aerosol challenge.
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Kafai NM, Williamson LE, Binshtein E, Sukupolvi-Petty S, Gardner CL, Liu J, Mackin S, Kim AS, Kose N, Carnahan RH, Jung A, Droit L, Reed DS, Handley SA, Klimstra WB, Crowe JE, and Diamond MS
- Subjects
- Aerosols, Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, Cryoelectron Microscopy, Horses, Mice, Encephalitis Virus, Venezuelan Equine, Encephalomyelitis, Venezuelan Equine prevention & control, Viral Vaccines
- Abstract
Venezuelan equine encephalitis virus (VEEV) remains a risk for epidemic emergence or use as an aerosolized bioweapon. To develop possible countermeasures, we isolated VEEV-specific neutralizing monoclonal antibodies (mAbs) from mice and a human immunized with attenuated VEEV strains. Functional assays and epitope mapping established that potently inhibitory anti-VEEV mAbs bind distinct antigenic sites in the A or B domains of the E2 glycoprotein and block multiple steps in the viral replication cycle including attachment, fusion, and egress. A 3.2-Å cryo-electron microscopy reconstruction of VEEV virus-like particles bound by a human Fab suggests that antibody engagement of the B domain may result in cross-linking of neighboring spikes to prevent conformational requirements for viral fusion. Prophylaxis or postexposure therapy with these mAbs protected mice against lethal aerosol challenge with VEEV. Our study defines functional and structural mechanisms of mAb protection and suggests that multiple antigenic determinants on VEEV can be targeted for vaccine or antibody-based therapeutic development., Competing Interests: Disclosures: M.S. Diamond is a consultant for Inbios, Vir Biotechnology, and Carnival Corporation, and on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory has received funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. Washington University has licensed murine anti-VEEV mAbs, and M.S. Diamond, S. Sukupolvi-Petty, and N.M. Kafai are inventors. J.E. Crowe has served as a consultant for Luna Biologics, is a member of the Scientific Advisory Boards of Meissa Vaccines, and is founder of IDBiologics. The Crowe laboratory has received unrelated sponsored research agreements from Takeda, AstraZeneca, and IDBiologics. J.E. Crowe reported grants from NIH during the conduct of the study and from GSK and Merck and grants from IDBiologics outside the submitted work; in addition, J. Crowe had a patent app for human antibodies for VEEV pending. The Klimstra laboratory has received unrelated research support from SAB Biotherapeutics and Tiba Biotechnology. The other authors have no additional conflicts of interest to declare. No other disclosures were reported., (© 2022 Kafai et al.)
- Published
- 2022
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16. Genetic and structural basis for SARS-CoV-2 variant neutralization by a two-antibody cocktail.
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Dong J, Zost SJ, Greaney AJ, Starr TN, Dingens AS, Chen EC, Chen RE, Case JB, Sutton RE, Gilchuk P, Rodriguez J, Armstrong E, Gainza C, Nargi RS, Binshtein E, Xie X, Zhang X, Shi PY, Logue J, Weston S, McGrath ME, Frieman MB, Brady T, Tuffy KM, Bright H, Loo YM, McTamney PM, Esser MT, Carnahan RH, Diamond MS, Bloom JD, and Crowe JE Jr
- Subjects
- Antibodies, Monoclonal chemistry, Antibodies, Monoclonal genetics, Antibodies, Monoclonal immunology, Antibodies, Neutralizing immunology, Antibodies, Viral chemistry, Antibodies, Viral genetics, Antibodies, Viral immunology, Antigenic Variation, Binding Sites, COVID-19 immunology, COVID-19 virology, Complementarity Determining Regions chemistry, Complementarity Determining Regions genetics, Humans, Mutation, Protein Domains, Spike Glycoprotein, Coronavirus chemistry, Spike Glycoprotein, Coronavirus genetics, Spike Glycoprotein, Coronavirus immunology, Antibodies, Neutralizing chemistry, Antibodies, Neutralizing genetics, SARS-CoV-2 immunology
- Abstract
Understanding the molecular basis for immune recognition of SARS-CoV-2 spike glycoprotein antigenic sites will inform the development of improved therapeutics. We determined the structures of two human monoclonal antibodies-AZD8895 and AZD1061-which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor-binding domain (RBD) of SARS-CoV-2 to define the genetic and structural basis of neutralization. AZD8895 forms an 'aromatic cage' at the heavy/light chain interface using germ line-encoded residues in complementarity-determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals. AZD1061 has an unusually long LCDR1; the HCDR3 makes interactions with the opposite face of the RBD from that of AZD8895. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the crucial binding residues of both antibodies and identified positions of concern with regards to virus escape from antibody-mediated neutralization. Both AZD8895 and AZD1061 have strong neutralizing activity against SARS-CoV-2 and variants of concern with antigenic substitutions in the RBD. We conclude that germ line-encoded antibody features enable recognition of the SARS-CoV-2 spike RBD and demonstrate the utility of the cocktail AZD7442 in neutralizing emerging variant viruses., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)
- Published
- 2021
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17. Cooperativity mediated by rationally selected combinations of human monoclonal antibodies targeting the henipavirus receptor binding protein.
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Doyle MP, Kose N, Borisevich V, Binshtein E, Amaya M, Nagel M, Annand EJ, Armstrong E, Bombardi R, Dong J, Schey KL, Broder CC, Zeitlin L, Kuang EA, Bornholdt ZA, West BR, Geisbert TW, Cross RW, and Crowe JE Jr
- Subjects
- Animals, Antibody Specificity, Chlorocebus aethiops, Cross Reactions, Disease Models, Animal, Drug Therapy, Combination, Ephrin-B2 immunology, Ephrin-B2 metabolism, Ephrin-B3 immunology, Ephrin-B3 metabolism, Female, Henipavirus Infections immunology, Henipavirus Infections metabolism, Henipavirus Infections virology, Host-Pathogen Interactions, Humans, Mesocricetus, Receptors, Virus immunology, Receptors, Virus metabolism, Vero Cells, Antibodies, Monoclonal pharmacology, Antibodies, Neutralizing pharmacology, Antiviral Agents pharmacology, Ephrin-B2 antagonists & inhibitors, Ephrin-B3 antagonists & inhibitors, Henipavirus pathogenicity, Henipavirus Infections prevention & control, Receptors, Virus antagonists & inhibitors
- Abstract
Hendra virus and Nipah virus (NiV), members of the Henipavirus (HNV) genus, are zoonotic paramyxoviruses known to cause severe disease across six mammalian orders, including humans. We isolated a panel of human monoclonal antibodies (mAbs) from the B cells of an individual with prior exposure to equine Hendra virus (HeV) vaccine, targeting distinct antigenic sites. The most potent class of cross-reactive antibodies achieves neutralization by blocking viral attachment to the host cell receptors ephrin-B2 and ephrin-B3, with a second class being enhanced by receptor binding. mAbs from both classes display synergistic activity in vitro. In a stringent hamster model of NiV Bangladesh (NiV
B ) infection, antibodies from both classes reduce morbidity and mortality and achieve synergistic protection in combination. These candidate mAbs might be suitable for use in a cocktail therapeutic approach to achieve synergistic potency and reduce the risk of virus escape., Competing Interests: Declaration of interests J.E.C. has served as a consultant for Luna Biologics, is a member of the Scientific Advisory Board of Meissa Vaccines, and is Founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received unrelated sponsored research agreements from Takeda, IDBiologics, and AstraZeneca. E.A.K., Z.A.B., and B.R.W. are employees and shareholders of Mapp. L.Z. is an employee, shareholder, and co-owner of Mapp. C.C.B. is a US federal employee, and C.C.B. and M.A. are co-inventors on US and foreign patents pertaining to Cedar virus and methods of use and recombinant Cedar virus chimeras, whose assignees are the US as represented by the Henry M. Jackson Foundation for the Advancement of Military Medicine (Bethesda, MD, USA). The remaining authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2021
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18. Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals.
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Chen EC, Gilchuk P, Zost SJ, Suryadevara N, Winkler ES, Cabel CR, Binshtein E, Chen RE, Sutton RE, Rodriguez J, Day S, Myers L, Trivette A, Williams JK, Davidson E, Li S, Doranz BJ, Campos SK, Carnahan RH, Thorne CA, Diamond MS, and Crowe JE Jr
- Abstract
Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases, as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identify 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, angiotensin-converting enzyme 2 [ACE2]-blocking clone that protects in vivo) and others recognizing non-RBD epitopes that bind the S2 domain. Germline-revertant forms of some public clonotypes bind efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants., Competing Interests: Declaration of interests E.D., J.K.W., and B.J.D. are employees of Integral Molecular, and B.J.D. is a shareholder in that company. M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, and Carnival Corporation and is on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory has received funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. J.E.C. has served as a consultant for Luna Biologics, is a member of the Scientific Advisory Board of Meissa Vaccines and is Founder of IDBiologics. The Crowe laboratory has received funding support in sponsored research agreements from AstraZeneca, IDBiologics, and Takeda., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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19. Neutralizing and protective human monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein.
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Suryadevara N, Shrihari S, Gilchuk P, VanBlargan LA, Binshtein E, Zost SJ, Nargi RS, Sutton RE, Winkler ES, Chen EC, Fouch ME, Davidson E, Doranz BJ, Chen RE, Shi PY, Carnahan RH, Thackray LB, Diamond MS, and Crowe JE Jr
- Subjects
- Animals, Binding, Competitive, COVID-19 immunology, COVID-19 virology, Chemokines metabolism, Chlorocebus aethiops, HEK293 Cells, Humans, Immunoglobulin Fab Fragments metabolism, Immunoglobulin G metabolism, Lung metabolism, Mice, Inbred C57BL, Models, Molecular, Mutagenesis genetics, Neutralization Tests, Protein Domains, Vero Cells, Mice, Antibodies, Monoclonal pharmacology, Antibodies, Neutralizing pharmacology, Protective Agents pharmacology, Spike Glycoprotein, Coronavirus chemistry, Spike Glycoprotein, Coronavirus immunology
- Abstract
Most human monoclonal antibodies (mAbs) neutralizing SARS-CoV-2 recognize the spike (S) protein receptor-binding domain and block virus interactions with the cellular receptor angiotensin-converting enzyme 2. We describe a panel of human mAbs binding to diverse epitopes on the N-terminal domain (NTD) of S protein from SARS-CoV-2 convalescent donors and found a minority of these possessed neutralizing activity. Two mAbs (COV2-2676 and COV2-2489) inhibited infection of authentic SARS-CoV-2 and recombinant VSV/SARS-CoV-2 viruses. We mapped their binding epitopes by alanine-scanning mutagenesis and selection of functional SARS-CoV-2 S neutralization escape variants. Mechanistic studies showed that these antibodies neutralize in part by inhibiting a post-attachment step in the infection cycle. COV2-2676 and COV2-2489 offered protection either as prophylaxis or therapy, and Fc effector functions were required for optimal protection. Thus, natural infection induces a subset of potent NTD-specific mAbs that leverage neutralizing and Fc-mediated activities to protect against SARS-CoV-2 infection using multiple functional attributes., Competing Interests: Declaration of interests M.E.F., E.D., and B.J.D. are employees of Integral Molecular. B.J.D. is a shareholder of Integral Molecular. M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, and Carnival Corporation and is on the Scientific Advisory Boards of Moderna and Immunome, and the Diamond laboratory at Washington University School of Medicine has received sponsored research agreements from Emergent BioSolutions, Moderna, and Vir Biotechnology. J.E.C. has served as a consultant for Eli Lilly, GlaxoSmithKline, and Luna Biologics, is a member of the Scientific Advisory Boards of CompuVax and Meissa Vaccines, and is founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received unrelated sponsored research agreements from IDBiologics and AstraZeneca., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
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20. Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-antibody cocktail.
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Dong J, Zost S, Greaney A, Starr TN, Dingens AS, Chen EC, Chen R, Case B, Sutton R, Gilchuk P, Rodriguez J, Armstrong E, Gainza C, Nargi R, Binshtein E, Xie X, Zhang X, Shi PY, Logue J, Weston S, McGrath M, Frieman M, Brady T, Tuffy K, Bright H, Loo YM, McvTamney P, Esser M, Carnahan R, Diamond M, Bloom J, and Crowe JE
- Abstract
The SARS-CoV-2 pandemic has led to an urgent need to understand the molecular basis for immune recognition of SARS-CoV-2 spike (S) glycoprotein antigenic sites. To define the genetic and structural basis for SARS-CoV-2 neutralization, we determined the structures of two human monoclonal antibodies COV2-2196 and COV2-21301, which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor binding domain (RBD) of SARS-CoV-2. COV2-2196 forms an 'aromatic cage' at the heavy/light chain interface using germline-encoded residues in complementarity determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals1-4. The structure of COV2-2130 reveals that an unusually long LCDR1 and HCDR3 make interactions with the opposite face of the RBD from that of COV2-2196. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the critical residues of both antibodies and identified positions of concern for possible viral escape. Nonetheless, both COV2-2196 and COV2130 showed strong neutralizing activity against SARS-CoV-2 strain with recent variations of concern including E484K, N501Y, and D614G substitutions. These studies reveal germline-encoded antibody features enabling recognition of the RBD and demonstrate the activity of a cocktail like AZD7442 in preventing escape from emerging variant viruses.
- Published
- 2021
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21. Complete Mapping of Mutations to the SARS-CoV-2 Spike Receptor-Binding Domain that Escape Antibody Recognition.
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Greaney AJ, Starr TN, Gilchuk P, Zost SJ, Binshtein E, Loes AN, Hilton SK, Huddleston J, Eguia R, Crawford KHD, Dingens AS, Nargi RS, Sutton RE, Suryadevara N, Rothlauf PW, Liu Z, Whelan SPJ, Carnahan RH, Crowe JE Jr, and Bloom JD
- Subjects
- Angiotensin-Converting Enzyme 2 chemistry, Angiotensin-Converting Enzyme 2 metabolism, Antibodies, Monoclonal immunology, Antibodies, Neutralizing immunology, Binding Sites, Epitopes immunology, Gene Library, High-Throughput Nucleotide Sequencing, Humans, Protein Domains, SARS-CoV-2 genetics, Saccharomyces cerevisiae genetics, Spike Glycoprotein, Coronavirus chemistry, SARS-CoV-2 immunology, Spike Glycoprotein, Coronavirus genetics, Spike Glycoprotein, Coronavirus immunology, Spike Glycoprotein, Coronavirus metabolism
- Abstract
Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and are a major contributor to neutralizing antibody responses elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies. They further enable the design of escape-resistant antibody cocktails-including cocktails of antibodies that compete for binding to the same RBD surface but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution., Competing Interests: Declaration of Interests J.E.C. has served as a consultant for Sanofi, is on the Scientific Advisory Boards of CompuVax and Meissa Vaccines, is a recipient of previous unrelated grants from Moderna and Sanofi, and is a founder of IDBiologics. Vanderbilt University has applied for patents on SARS-CoV-2 antibodies. S.P.J.W. and P.W.R. have filed a disclosure with Washington University for recombinant VSV. The other authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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22. Human Antibodies Protect against Aerosolized Eastern Equine Encephalitis Virus Infection.
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Williamson LE, Gilliland T Jr, Yadav PK, Binshtein E, Bombardi R, Kose N, Nargi RS, Sutton RE, Durie CL, Armstrong E, Carnahan RH, Walker LM, Kim AS, Fox JM, Diamond MS, Ohi MD, Klimstra WB, and Crowe JE Jr
- Subjects
- Adult, Animals, Antibodies, Monoclonal isolation & purification, Antibodies, Neutralizing immunology, Antigens, Viral immunology, Cryoelectron Microscopy, Disease Models, Animal, Encephalitis Virus, Eastern Equine ultrastructure, Encephalomyelitis, Equine virology, Epitopes chemistry, Female, Glycoproteins immunology, Humans, Mice, Models, Molecular, Mutagenesis genetics, Neutralization Tests, Protein Binding, Protein Domains, Recombinant Proteins immunology, Sindbis Virus immunology, Virion immunology, Virion ultrastructure, Virus Internalization, Aerosols administration & dosage, Antibodies, Monoclonal immunology, Antibodies, Viral immunology, Encephalitis Virus, Eastern Equine immunology, Encephalomyelitis, Equine immunology, Encephalomyelitis, Equine prevention & control
- Abstract
Eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has recently shown an increase in human cases. Here, we characterize human monoclonal antibodies (mAbs) isolated from a survivor of natural EEEV infection with potent (<20 pM) inhibitory activity of EEEV. Cryo-electron microscopy reconstructions of two highly neutralizing mAbs, EEEV-33 and EEEV-143, were solved in complex with chimeric Sindbis/EEEV virions to 7.2 Å and 8.3 Å, respectively. The mAbs recognize two distinct antigenic sites that are critical for inhibiting viral entry into cells. EEEV-33 and EEEV-143 protect against disease following stringent lethal aerosol challenge of mice with highly pathogenic EEEV. These studies provide insight into the molecular basis for the neutralizing human antibody response against EEEV and can facilitate development of vaccines and candidate antibody therapeutics., Competing Interests: Declaration of Interests M.S.D. is consultant for Inbios, Vir Biotechnology, and NGM Biopharmaceuticals, is on the Scientific Advisory Boards of Moderna and Immunome, and is a recipient of grants from Moderna, Vir Biotechnology, and Emergent BioSolutions. J.E.C. has served as consultant for Eli Lilly and Sanofi, is on Scientific Advisory Boards of CompuVax and Meissa Vaccines, is recipient of research grants from Moderna and Sanofi, and is Founder of IDBiologics, Inc. Vanderbilt University has applied for patents that are related to this work., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
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23. Structure and assembly of CAV1 8S complexes revealed by single particle electron microscopy.
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Han B, Porta JC, Hanks JL, Peskova Y, Binshtein E, Dryden K, Claxton DP, Mchaourab HS, Karakas E, Ohi MD, and Kenworthy AK
- Abstract
Highly stable oligomeric complexes of the monotopic membrane protein caveolin serve as fundamental building blocks of caveolae. Current evidence suggests these complexes are disc shaped, but the details of their structural organization and how they assemble are poorly understood. Here, we address these questions using single particle electron microscopy of negatively stained recombinant 8S complexes of human caveolin 1. We show that 8S complexes are toroidal structures ~15 nm in diameter that consist of an outer ring, an inner ring, and central protruding stalk. Moreover, we map the position of the N and C termini and determine their role in complex assembly, and visualize the 8S complexes in heterologous caveolae. Our findings provide critical insights into the structural features of 8S complexes and allow us to propose a model for how these highly stable membrane-embedded complexes are generated., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)
- Published
- 2020
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24. Complete mapping of mutations to the SARS-CoV-2 spike receptor-binding domain that escape antibody recognition.
- Author
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Greaney AJ, Starr TN, Gilchuk P, Zost SJ, Binshtein E, Loes AN, Hilton SK, Huddleston J, Eguia R, Crawford KHD, Dingens AS, Nargi RS, Sutton RE, Suryadevara N, Rothlauf PW, Liu Z, Whelan SPJ, Carnahan RH, Crowe JE Jr, and Bloom JD
- Abstract
Antibodies targeting the SARS-CoV-2 spike receptor-binding domain (RBD) are being developed as therapeutics and make a major contribution to the neutralizing antibody response elicited by infection. Here, we describe a deep mutational scanning method to map how all amino-acid mutations in the RBD affect antibody binding, and apply this method to 10 human monoclonal antibodies. The escape mutations cluster on several surfaces of the RBD that broadly correspond to structurally defined antibody epitopes. However, even antibodies targeting the same RBD surface often have distinct escape mutations. The complete escape maps predict which mutations are selected during viral growth in the presence of single antibodies, and enable us to design escape-resistant antibody cocktails-including cocktails of antibodies that compete for binding to the same surface of the RBD but have different escape mutations. Therefore, complete escape-mutation maps enable rational design of antibody therapeutics and assessment of the antigenic consequences of viral evolution., Competing Interests: Declarations of Interests J.E.C. has served as a consultant for Sanofi; is on the Scientific Advisory Boards of CompuVax and Meissa Vaccines; is a recipient of previous unrelated research grants from Moderna and Sanofi; and is a founder of IDBiologics. Vanderbilt University has applied for patents concerning SARS-CoV-2 antibodies analyzed in this work. S.P.J.W. and P.W.R. have filed a disclosure with Washington University for the recombinant VSV. The other authors declare no competing interests.
- Published
- 2020
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25. Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein.
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Zost SJ, Gilchuk P, Chen RE, Case JB, Reidy JX, Trivette A, Nargi RS, Sutton RE, Suryadevara N, Chen EC, Binshtein E, Shrihari S, Ostrowski M, Chu HY, Didier JE, MacRenaris KW, Jones T, Day S, Myers L, Eun-Hyung Lee F, Nguyen DC, Sanz I, Martinez DR, Rothlauf PW, Bloyet LM, Whelan SPJ, Baric RS, Thackray LB, Diamond MS, Carnahan RH, and Crowe JE Jr
- Subjects
- Antibodies, Monoclonal immunology, Antibodies, Monoclonal therapeutic use, Antibodies, Neutralizing immunology, Antibodies, Neutralizing isolation & purification, Betacoronavirus immunology, Betacoronavirus pathogenicity, COVID-19, Coronavirus Infections immunology, Coronavirus Infections virology, Humans, Pandemics, Pneumonia, Viral immunology, Pneumonia, Viral virology, Protein Binding, SARS-CoV-2, Spike Glycoprotein, Coronavirus immunology, Antibodies, Monoclonal isolation & purification, Betacoronavirus drug effects, Coronavirus Infections drug therapy, Pneumonia, Viral drug therapy, Spike Glycoprotein, Coronavirus antagonists & inhibitors
- Abstract
Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date
1,2 . In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes on the basis of their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of advanced antibody discovery platforms.- Published
- 2020
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26. Potently neutralizing and protective human antibodies against SARS-CoV-2.
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Zost SJ, Gilchuk P, Case JB, Binshtein E, Chen RE, Nkolola JP, Schäfer A, Reidy JX, Trivette A, Nargi RS, Sutton RE, Suryadevara N, Martinez DR, Williamson LE, Chen EC, Jones T, Day S, Myers L, Hassan AO, Kafai NM, Winkler ES, Fox JM, Shrihari S, Mueller BK, Meiler J, Chandrashekar A, Mercado NB, Steinhardt JJ, Ren K, Loo YM, Kallewaard NL, McCune BT, Keeler SP, Holtzman MJ, Barouch DH, Gralinski LE, Baric RS, Thackray LB, Diamond MS, Carnahan RH, and Crowe JE Jr
- Subjects
- Angiotensin-Converting Enzyme 2, Animals, Antibodies, Monoclonal immunology, Betacoronavirus chemistry, Binding, Competitive, COVID-19, Cell Line, Cross Reactions, Disease Models, Animal, Epitopes, B-Lymphocyte chemistry, Epitopes, B-Lymphocyte immunology, Female, Humans, Macaca mulatta, Male, Mice, Middle Aged, Neutralization Tests, Peptidyl-Dipeptidase A genetics, Peptidyl-Dipeptidase A metabolism, Pre-Exposure Prophylaxis, Severe acute respiratory syndrome-related coronavirus chemistry, Severe acute respiratory syndrome-related coronavirus immunology, SARS-CoV-2, Severe Acute Respiratory Syndrome immunology, Spike Glycoprotein, Coronavirus chemistry, Spike Glycoprotein, Coronavirus immunology, Spike Glycoprotein, Coronavirus metabolism, Antibodies, Neutralizing immunology, Antibodies, Viral immunology, Betacoronavirus immunology, Coronavirus Infections immunology, Coronavirus Infections prevention & control, Pandemics prevention & control, Pneumonia, Viral immunology, Pneumonia, Viral prevention & control
- Abstract
The ongoing pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health
1 and the medical countermeasures available so far are limited2,3 . Moreover, we currently lack a thorough understanding of the mechanisms of humoral immunity to SARS-CoV-24 . Here we analyse a large panel of human monoclonal antibodies that target the spike (S) glycoprotein5 , and identify several that exhibit potent neutralizing activity and fully block the receptor-binding domain of the S protein (SRBD ) from interacting with human angiotensin-converting enzyme 2 (ACE2). Using competition-binding, structural and functional studies, we show that the monoclonal antibodies can be clustered into classes that recognize distinct epitopes on the SRBD , as well as distinct conformational states of the S trimer. Two potently neutralizing monoclonal antibodies, COV2-2196 and COV2-2130, which recognize non-overlapping sites, bound simultaneously to the S protein and neutralized wild-type SARS-CoV-2 virus in a synergistic manner. In two mouse models of SARS-CoV-2 infection, passive transfer of COV2-2196, COV2-2130 or a combination of both of these antibodies protected mice from weight loss and reduced the viral burden and levels of inflammation in the lungs. In addition, passive transfer of either of two of the most potent ACE2-blocking monoclonal antibodies (COV2-2196 or COV2-2381) as monotherapy protected rhesus macaques from SARS-CoV-2 infection. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic agents.- Published
- 2020
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27. Potently neutralizing human antibodies that block SARS-CoV-2 receptor binding and protect animals.
- Author
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Zost SJ, Gilchuk P, Case JB, Binshtein E, Chen RE, Reidy JX, Trivette A, Nargi RS, Sutton RE, Suryadevara N, Williamson LE, Chen EC, Jones T, Day S, Myers L, Hassan AO, Kafai NM, Winkler ES, Fox JM, Steinhardt JJ, Ren K, Loo YM, Kallewaard NL, Martinez DR, Schäfer A, Gralinski LE, Baric RS, Thackray LB, Diamond MS, Carnahan RH, and Crowe JE
- Abstract
The COVID-19 pandemic is a major threat to global health for which there are only limited medical countermeasures, and we lack a thorough understanding of mechanisms of humoral immunity
1,2 . From a panel of monoclonal antibodies (mAbs) targeting the spike (S) glycoprotein isolated from the B cells of infected subjects, we identified several mAbs that exhibited potent neutralizing activity with IC50 values as low as 0.9 or 15 ng/mL in pseudovirus or wild-type ( wt ) SARS-CoV-2 neutralization tests, respectively. The most potent mAbs fully block the receptor-binding domain of S (SRBD ) from interacting with human ACE2. Competition-binding, structural, and functional studies allowed clustering of the mAbs into defined classes recognizing distinct epitopes within major antigenic sites on the SRBD . Electron microscopy studies revealed that these mAbs recognize distinct conformational states of trimeric S protein. Potent neutralizing mAbs recognizing unique sites, COV2-2196 and COV2-2130, bound simultaneously to S and synergistically neutralized authentic SARS-CoV-2 virus. In two murine models of SARS-CoV-2 infection, passive transfer of either COV2-2916 or COV2-2130 alone or a combination of both mAbs protected mice from severe weight loss and reduced viral burden and inflammation in the lung. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic cocktails.- Published
- 2020
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28. Mammalian Retromer Is an Adaptable Scaffold for Cargo Sorting from Endosomes.
- Author
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Kendall AK, Xie B, Xu P, Wang J, Burcham R, Frazier MN, Binshtein E, Wei H, Graham TR, Nakagawa T, and Jackson LP
- Subjects
- Animals, Cryoelectron Microscopy, Humans, Mice, Mutation, Protein Domains, Protein Transport, Saccharomyces cerevisiae, Vesicular Transport Proteins genetics, Vesicular Transport Proteins metabolism, Endosomes metabolism, Protein Multimerization, Vesicular Transport Proteins chemistry
- Abstract
Metazoan retromer (VPS26/VPS35/VPS29) associates with sorting nexins on endosomal tubules to sort proteins to the trans-Golgi network or plasma membrane. Mechanisms of metazoan retromer assembly remain undefined. We combine single-particle cryoelectron microscopy with biophysical methods to uncover multiple oligomer structures. 2D class averages reveal mammalian heterotrimers; dimers of trimers; tetramers of trimers; and flat chains. These species are further supported by biophysical solution studies. We provide reconstructions of all species, including key sub-structures (∼5 Å resolution). Local resolution variation suggests that heterotrimers and dimers adopt multiple conformations. Our structures identify a flexible, highly conserved electrostatic dimeric interface formed by VPS35 subunits. We generate structure-based mutants to disrupt this interface in vitro. Equivalent mutations in yeast demonstrate a mild cargo-sorting defect. Our data suggest the metazoan retromer is an adaptable and plastic scaffold that accommodates interactions with different sorting nexins to sort multiple cargoes from endosomes their final destinations., Competing Interests: Declaration of Interests The authors have no competing interests to declare., (Copyright © 2020 Elsevier Ltd. All rights reserved.)
- Published
- 2020
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29. Multifunctional Pan-ebolavirus Antibody Recognizes a Site of Broad Vulnerability on the Ebolavirus Glycoprotein.
- Author
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Gilchuk P, Kuzmina N, Ilinykh PA, Huang K, Gunn BM, Bryan A, Davidson E, Doranz BJ, Turner HL, Fusco ML, Bramble MS, Hoff NA, Binshtein E, Kose N, Flyak AI, Flinko R, Orlandi C, Carnahan R, Parrish EH, Sevy AM, Bombardi RG, Singh PK, Mukadi P, Muyembe-Tamfum JJ, Ohi MD, Saphire EO, Lewis GK, Alter G, Ward AB, Rimoin AW, Bukreyev A, and Crowe JE Jr
- Subjects
- 3T3 Cells, Adult, Animals, CHO Cells, Cell Line, Chlorocebus aethiops, Cricetulus, Disease Models, Animal, Drosophila, Female, Ferrets, Guinea Pigs, Hemorrhagic Fever, Ebola prevention & control, Hemorrhagic Fever, Ebola virology, Humans, Immunoglobulin G immunology, Jurkat Cells, Male, Mice, Mice, Inbred BALB C, Mice, Knockout, THP-1 Cells, Vero Cells, Antibodies, Monoclonal immunology, Antibodies, Monoclonal pharmacology, Antibodies, Neutralizing immunology, Antibodies, Neutralizing pharmacology, Antibodies, Viral immunology, Antibodies, Viral pharmacology, Ebolavirus immunology, Glycoproteins immunology, Hemorrhagic Fever, Ebola immunology
- Abstract
Ebolaviruses cause severe disease in humans, and identification of monoclonal antibodies (mAbs) that are effective against multiple ebolaviruses are important for therapeutics development. Here we describe a distinct class of broadly neutralizing human mAbs with protective capacity against three ebolaviruses infectious for humans: Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) viruses. We isolated mAbs from human survivors of ebolavirus disease and identified a potent mAb, EBOV-520, which bound to an epitope in the glycoprotein (GP) base region. EBOV-520 efficiently neutralized EBOV, BDBV, and SUDV and also showed protective capacity in relevant animal models of these infections. EBOV-520 mediated protection principally by direct virus neutralization and exhibited multifunctional properties. This study identified a potent naturally occurring mAb and defined key features of the human antibody response that may contribute to broad protection. This multifunctional mAb and related clones are promising candidates for development as broadly protective pan-ebolavirus therapeutic molecules., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
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30. Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.
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Mousa JJ, Binshtein E, Human S, Fong RH, Alvarado G, Doranz BJ, Moore ML, Ohi MD, and Crowe JE Jr
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Antibodies, Monoclonal chemistry, Antibodies, Neutralizing chemistry, Antibodies, Viral chemistry, Antibody Specificity, Binding Sites, Antibody, Binding, Competitive, Cross Reactions, Epitope Mapping, Humans, Kinetics, Metapneumovirus immunology, Metapneumovirus metabolism, Microscopy, Electron, Mutation, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Respiratory Syncytial Virus, Human immunology, Viral Fusion Proteins antagonists & inhibitors, Viral Fusion Proteins genetics, Antibodies, Monoclonal metabolism, Antibodies, Neutralizing metabolism, Antibodies, Viral metabolism, Epitopes metabolism, Respiratory Syncytial Virus, Human metabolism, Viral Fusion Proteins metabolism
- Abstract
Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.
- Published
- 2018
- Full Text
- View/download PDF
31. While the revolution will not be crystallized, biochemistry reigns supreme.
- Author
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Takizawa Y, Binshtein E, Erwin AL, Pyburn TM, Mittendorf KF, and Ohi MD
- Subjects
- Crystallography, X-Ray methods, Cryoelectron Microscopy methods, Models, Molecular
- Abstract
Single-particle cryo-electron microscopy (EM) is currently gaining attention for the ability to calculate structures that reach sub-5 Å resolutions; however, the technique is more than just an alternative approach to X-ray crystallography. Molecular machines work via dynamic conformational changes, making structural flexibility the hallmark of function. While the dynamic regions in molecules are essential, they are also the most challenging to structurally characterize. Single-particle EM has the distinct advantage of being able to directly visualize purified molecules without the formation of ordered arrays of molecules locked into identical conformations. Additionally, structures determined using single-particle EM can span resolution ranges from very low- to atomic-levels (>30-1.8 Å), sometimes even in the same structure. The ability to accommodate various resolutions gives single-particle EM the unique capacity to structurally characterize dynamic regions of biological molecules, thereby contributing essential structural information needed for the development of molecular models that explain function. Further, many important molecular machines are intrinsically dynamic and compositionally heterogeneous. Structures of these complexes may never reach sub-5 Å resolutions due to this flexibility required for function. Thus, the biochemical quality of the sample, as well as, the calculation and interpretation of low- to mid-resolution cryo-EM structures (30-8 Å) remains critical for generating insights into the architecture of many challenging biological samples that cannot be visualized using alternative techniques., (© 2016 The Protein Society.)
- Published
- 2017
- Full Text
- View/download PDF
32. Reducing the specific energy consumption of 1st-pass SWRO by application of high-flux membranes fed with high-pH, decarbonated seawater.
- Author
-
Ophek L, Birnhack L, Nir O, Binshtein E, and Lahav O
- Subjects
- Boron chemistry, Filtration economics, Hydrogen-Ion Concentration, Membranes, Artificial, Osmosis, Water Purification economics, Filtration methods, Seawater chemistry, Water Purification methods
- Abstract
A new operational approach is presented, which has the potential to substantially cut down on the energy and cost demand associated with seawater reverse osmosis (SWRO) desalination, without changing the currently-installed infrastructure. The approach comprises acidification/decarbonation of the feed seawater followed by high-pH single RO pass using high-flux membranes. Since the limitation imposed by CaCO3(s) precipitation is overcome, the recovery ratio can be significantly increased. This work presents a new operational concept aimed at maximizing the benefits that can be obtained from new low-energy RO membranes available on the market. Results obtained from operating a pilot RO system revealed that following an acidification and decarbonation step, recovery ratio of 56% could be practically attained, along with effluent TDS and boron concentrations of 375 and 0.3 mg/l, respectively (feed water pH was adjusted to pH9.53 following the decarbonation step). The specific energy consumption (SEC) of this operation was calculated to be 5%-10% lower than the SEC typically associated with "conventional" SWRO operation. Two further scenarios were theoretically considered, under which the limiting operational parameter became Mg(OH)2(s) and BaSO4(s) precipitation. It was concluded that despite the fact that higher recovery ratios could be obtained, the high pressure required in these scenarios made them less appealing from both the SEC and cost standpoints. The normalized cost of the suggested approach was found to be ∼$0.07 ± 0.02/m(3) cheaper than the currently-practiced SWRO approach for obtaining product water characterized by TDS < 500 and B < 0.5 mg/l., (Copyright © 2015 Elsevier Ltd. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
33. Cryo-electron microscopy and the amazing race to atomic resolution.
- Author
-
Binshtein E and Ohi MD
- Subjects
- Computer Simulation, Cryoelectron Microscopy instrumentation, Models, Molecular, Protein Conformation, Proteins chemistry, Proteins ultrastructure, Cryoelectron Microscopy methods
- Abstract
Cryo-electron microscopy (cryo-EM), the structural analysis of samples embedded in vitreous ice, is a powerful approach for determining three-dimensional (3D) structures of biological specimens. Over the past two decades, this technique has been used to successfully calculate subnanometer (<10 Å) resolution and, in some cases, near-atomic resolution structures of highly symmetrical and stable complexes such as icosahedral viruses and ribosomes, as well as samples that form ordered two-dimensional or helical arrays. However, determining high-resolution 3D structures of smaller, less symmetrical, and dynamic samples remains a significant challenge. The recent development of electron microscopes with automated data collection capabilities and robust direct electron detection cameras, as well as new powerful image processing algorithms, has dramatically expanded the number of biological macromolecules amenable for study using cryo-EM. In addition, these new technological and computational developments have been used to successfully determine <5 Å resolution 3D structures of samples, such as membrane proteins and complexes with either low or no symmetry, that traditionally were not considered promising candidates for high-resolution cryo-EM. With these exciting new advances, cryo-EM is now on pace to determine atomic resolution 3D structures.
- Published
- 2015
- Full Text
- View/download PDF
34. Glyoxylate carboligase: a unique thiamin diphosphate-dependent enzyme that can cycle between the 4'-aminopyrimidinium and 1',4'-iminopyrimidine tautomeric forms in the absence of the conserved glutamate.
- Author
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Nemeria N, Binshtein E, Patel H, Balakrishnan A, Vered I, Shaanan B, Barak Z, Chipman D, and Jordan F
- Subjects
- Amino Acid Substitution, Circular Dichroism, Gene Expression Regulation, Hydrogen-Ion Concentration, Models, Molecular, Molecular Structure, Mutagenesis, Site-Directed, Pyruvic Acid, Substrate Specificity, Glutamic Acid chemistry, Ligases metabolism, Pyrimidines chemistry, Thiamine Pyrophosphate chemistry, Thiamine Pyrophosphate metabolism
- Abstract
Glyoxylate carboligase (GCL) is a thiamin diphosphate (ThDP)-dependent enzyme, which catalyzes the decarboxylation of glyoxylate and ligation to a second molecule of glyoxylate to form tartronate semialdehyde (TSA). This enzyme is unique among ThDP enzymes in that it lacks a conserved glutamate near the N1' atom of ThDP (replaced by Val51) or any other potential acid-base side chains near ThDP. The V51D substitution shifts the pH optimum to 6.0-6.2 (pK(a) of 6.2) for TSA formation from pH 7.0-7.7 in wild-type GCL. This pK(a) is similar to the pK(a) of 6.1 for the 1',4'-iminopyrimidine (IP)-4'-aminopyrimidinium (APH(+)) protonic equilibrium, suggesting that the same groups control both ThDP protonation and TSA formation. The key covalent ThDP-bound intermediates were identified on V51D GCL by a combination of steady-state and stopped-flow circular dichroism methods, yielding rate constants for their formation and decomposition. It was demonstrated that active center variants with substitution at I393 could synthesize (S)-acetolactate from pyruvate solely, and acetylglycolate derived from pyruvate as the acetyl donor and glyoxylate as the acceptor, implying that this substitutent favored pyruvate as the donor in carboligase reactions. Consistent with these observations, the I393A GLC variants could stabilize the predecarboxylation intermediate analogues derived from acetylphosphinate, propionylphosphinate, and methyl acetylphosphonate in their IP tautomeric forms notwithstanding the absence of the conserved glutamate. The role of the residue at the position occupied typically by the conserved Glu controls the pH dependence of kinetic parameters, while the entire reaction sequence could be catalyzed by ThDP itself, once the APH(+) form is accessible.
- Published
- 2012
- Full Text
- View/download PDF
35. Glyoxylate carboligase lacks the canonical active site glutamate of thiamine-dependent enzymes.
- Author
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Kaplun A, Binshtein E, Vyazmensky M, Steinmetz A, Barak Z, Chipman DM, Tittmann K, and Shaanan B
- Subjects
- Binding Sites, Carboxy-Lyases genetics, Carboxylic Acids chemistry, Carboxylic Acids metabolism, Circular Dichroism, Crystallography, X-Ray, Escherichia coli enzymology, Escherichia coli genetics, Kinetics, Models, Molecular, Mutation genetics, Phosphates chemistry, Protein Structure, Tertiary, Thiamine analogs & derivatives, Thiazoles chemistry, Thiazoles metabolism, Valine genetics, Valine metabolism, Carboxy-Lyases chemistry, Carboxy-Lyases metabolism, Glutamates chemistry, Glutamates metabolism, Thiamine chemistry, Thiamine metabolism
- Abstract
Thiamine diphosphate (ThDP), a derivative of vitamin B1, is an enzymatic cofactor whose special chemical properties allow it to play critical mechanistic roles in a number of essential metabolic enzymes. It has been assumed that all ThDP-dependent enzymes exploit a polar interaction between a strictly conserved glutamate and the N1' of the ThDP moiety. The crystal structure of glyoxylate carboligase challenges this paradigm by revealing that valine replaces the conserved glutamate. Through kinetic, spectroscopic and site-directed mutagenesis studies, we show that although this extreme change lowers the rate of the initial step of the enzymatic reaction, it ensures efficient progress through subsequent steps. Glyoxylate carboligase thus provides a unique illustration of the fine tuning between catalytic stages imposed during evolution on enzymes catalyzing multistep processes.
- Published
- 2008
- Full Text
- View/download PDF
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