28 results on '"Sophie M. C. Gobeil"'
Search Results
2. D614G Mutation Alters SARS-CoV-2 Spike Conformation and Enhances Protease Cleavage at the S1/S2 Junction
- Author
-
Sophie M.-C. Gobeil, Katarzyna Janowska, Shana McDowell, Katayoun Mansouri, Robert Parks, Kartik Manne, Victoria Stalls, Megan F. Kopp, Rory Henderson, Robert J. Edwards, Barton F. Haynes, and Priyamvada Acharya
- Subjects
COVID-19 ,SARS-CoV-2 ,spike ,2P ,D614G ,allostery ,Biology (General) ,QH301-705.5 - Abstract
Summary: The severe acute respiratory coronavirus 2 (SARS-CoV-2) spike (S) protein is the target of vaccine design efforts to end the coronavirus disease 2019 (COVID-19) pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic and are now the dominant form worldwide. Here, we explore S conformational changes and the effects of the D614G mutation on a soluble S ectodomain construct. Cryoelectron microscopy (cryo-EM) structures reveal altered receptor binding domain (RBD) disposition; antigenicity and proteolysis experiments reveal structural changes and enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage alters the up/down ratio of the RBDs in the G614 S ectodomain, demonstrating an allosteric effect on RBD positioning triggered by changes in the SD2 region, which harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 S conformational landscape and allostery and have implications for vaccine design.
- Published
- 2021
- Full Text
- View/download PDF
3. Cold sensitivity of the SARS-CoV-2 spike ectodomain
- Author
-
Megan Kopp, S. Munir Alam, Sophie M. C. Gobeil, R Parks, Priyamvada Acharya, Zekun Mu, Rory Henderson, Gregory D. Sempowski, Robert J. Edwards, Kartik Manne, Dapeng Li, Kevin O. Saunders, Jordan Sprenz, Katarzyna Janowska, Wilton B. Williams, Margaret Deyton, Xiaozhi Lu, Brian E. Watts, Barton F. Haynes, Thomas H. Oguin, Victoria Stalls, Katayoun Mansouri, and David C. Montefiori
- Subjects
Protein Denaturation ,2019-20 coronavirus outbreak ,COVID-19 Vaccines ,Coronavirus disease 2019 (COVID-19) ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Protein domain ,Enzyme-Linked Immunosorbent Assay ,Antibodies, Viral ,Article ,03 medical and health sciences ,0302 clinical medicine ,Protein stability ,Protein Domains ,Structural Biology ,medicine ,Humans ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Protein Stability ,Chemistry ,Cryoelectron Microscopy ,Surface Plasmon Resonance ,Cell biology ,Cold Temperature ,Ectodomain ,Spike Glycoprotein, Coronavirus ,Biophysics ,Cold sensitivity ,Spike (software development) ,medicine.symptom ,030217 neurology & neurosurgery - Abstract
The impact of COVID-19 and the urgency to develop a vaccine against the SARS-CoV-2 virus cannot be overstated. The viral fusion spike (S) protein ectodomain is the primary target for vaccine development. Here we report an unexpected cold sensitivity of a stabilized SARS-CoV-2 ectodomain construct currently being widely used for immunogen design. We found that when stored at 22 or 37 °C for 1 week, the S-protein displayed well-ordered trimeric spikes by negative stain electron microscopy. However, storage at 4 °C reduced the trimeric spikes to
- Published
- 2021
4. Cryo-EM structures of SARS-CoV-2 Omicron BA.2 spike
- Author
-
Victoria Stalls, Jared Lindenberger, Sophie M-C. Gobeil, Rory Henderson, Rob Parks, Maggie Barr, Margaret Deyton, Mitchell Martin, Katarzyna Janowska, Xiao Huang, Aaron May, Micah Speakman, Esther Beaudoin, Bryan Kraft, Xiaozhi Lu, Robert J Edwards, Amanda Eaton, David C. Montefiori, Wilton Williams, Kevin O. Saunders, Kevin Wiehe, Barton F. Haynes, and Priyamvada Acharya
- Subjects
SARS-CoV-2 ,Cryoelectron Microscopy ,Spike Glycoprotein, Coronavirus ,COVID-19 ,Humans ,Receptors, Virus ,Antibodies, Viral ,General Biochemistry, Genetics and Molecular Biology - Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2 sub-lineage has gained in proportion relative to BA.1. Because spike (S) protein variations may underlie differences in their pathobiology, here we determine cryoelectron microscopy (cryo-EM) structures of the BA.2 S ectodomain and compare these with previously determined BA.1 S structures. BA.2 receptor-binding domain (RBD) mutations induce remodeling of the RBD structure, resulting in tighter packing and improved thermostability. Interprotomer RBD interactions are enhanced in the closed (or 3-RBD-down) BA.2 S, while the fusion peptide is less accessible to antibodies than in BA.1. Binding and pseudovirus neutralization assays reveal extensive immune evasion while defining epitopes of two outer RBD face-binding antibodies, DH1044 and DH1193, that neutralize both BA.1 and BA.2. Taken together, our results indicate that stabilization of the closed state through interprotomer RBD-RBD packing is a hallmark of the Omicron variant and show differences in key functional regions in the BA.1 and BA.2 S proteins.
- Published
- 2022
5. Structural diversity of the SARS-CoV-2 Omicron spike
- Author
-
Sophie M-C. Gobeil, Rory Henderson, Victoria Stalls, Katarzyna Janowska, Xiao Huang, Aaron May, Micah Speakman, Esther Beaudoin, Kartik Manne, Dapeng Li, Rob Parks, Maggie Barr, Margaret Deyton, Mitchell Martin, Katayoun Mansouri, Robert J. Edwards, Gregory D. Sempowski, Kevin O. Saunders, Kevin Wiehe, Wilton Williams, Bette Korber, Barton F. Haynes, and Priyamvada Acharya
- Subjects
Article - Abstract
Aided by extensive spike protein mutation, the SARS-CoV-2 Omicron variant overtook the previously dominant Delta variant. Spike conformation plays an essential role in SARS-CoV-2 evolution via changes in receptor binding domain (RBD) and neutralizing antibody epitope presentation affecting virus transmissibility and immune evasion. Here, we determine cryo-EM structures of the Omicron and Delta spikes to understand the conformational impacts of mutations in each. The Omicron spike structure revealed an unusually tightly packed RBD organization with long range impacts that were not observed in the Delta spike. Binding and crystallography revealed increased flexibility at the functionally critical fusion peptide site in the Omicron spike. These results reveal a highly evolved Omicron spike architecture with possible impacts on its high levels of immune evasion and transmissibility.
- Published
- 2022
6. FKBP12 dimerization mutations effect FK506 binding and differentially alter calcineurin inhibition in the human pathogen Aspergillus fumigatus
- Author
-
Sophie M. C. Gobeil, D. Christopher Cole, William J. Steinbach, Joseph Heitman, Ronald A. Venters, Leonard D. Spicer, Praveen R. Juvvadi, and Benjamin G. Bobay
- Subjects
0301 basic medicine ,medicine.medical_treatment ,Calcineurin Inhibitors ,Mutant ,Biophysics ,Tacrolimus Binding Protein 1A ,medicine.disease_cause ,Biochemistry ,Article ,Protein Structure, Secondary ,Tacrolimus ,Aspergillus fumigatus ,Fungal Proteins ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Humans ,Computer Simulation ,Amino Acid Sequence ,Molecular Biology ,Gene ,Mutation ,biology ,Chemistry ,Calcineurin ,Active site ,Cell Biology ,biology.organism_classification ,Cell biology ,030104 developmental biology ,FKBP ,Immunosuppressive drug ,030220 oncology & carcinogenesis ,biology.protein ,Mutant Proteins ,Protein Multimerization ,Protein Binding - Abstract
The 12-kDa FK506-binding protein (FKBP12) is the target of the commonly used immunosuppressive drug FK506. The FKBP12-FK506 complex binds to calcineurin and inhibits its activity, leading to immunosuppression and preventing organ transplant rejection. Our recent characterization of crystal structures of FKBP12 proteins in pathogenic fungi revealed the involvement of the 80’s loop residue (Pro90) in the active site pocket in self-substrate interaction providing novel evidence on FKBP12 dimerization in vivo. The 40’s loop residues have also been shown to be involved in reversible dimerization of FKBP12 in the mammalian and yeast systems. To understand how FKBP12 dimerization affects FK506 binding and influences calcineurin function, we generated Aspergillus fumigatus FKBP12 mutations in the 40’s and 50’s loop (F37 M/L; W60V). Interestingly, the mutants exhibited variable FK506 susceptibility in vivo indicating differing dimer strengths. In comparison to the 80’s loop P90G and V91C mutants, the F37 M/L and W60V mutants exhibited greater FK506 resistance, with the F37M mutation showing complete loss in calcineurin binding in vivo. Molecular dynamics and pulling simulations for each dimeric FKBP12 protein revealed a two-fold increase in dimer strength and significantly higher number of contacts for the F37M, F37L, and W60V mutations, further confirming their varying degree of impact on FK506 binding and calcineurin inhibition in vivo.
- Published
- 2020
7. Structural diversity of the SARS-CoV-2 Omicron spike
- Author
-
Sophie M.-C. Gobeil, Rory Henderson, Victoria Stalls, Katarzyna Janowska, Xiao Huang, Aaron May, Micah Speakman, Esther Beaudoin, Kartik Manne, Dapeng Li, Rob Parks, Maggie Barr, Margaret Deyton, Mitchell Martin, Katayoun Mansouri, Robert J. Edwards, Amanda Eaton, David C. Montefiori, Gregory D. Sempowski, Kevin O. Saunders, Kevin Wiehe, Wilton Williams, Bette Korber, Barton F. Haynes, and Priyamvada Acharya
- Subjects
SARS-CoV-2 ,Mutation ,Spike Glycoprotein, Coronavirus ,COVID-19 ,Humans ,Cell Biology ,Angiotensin-Converting Enzyme 2 ,Molecular Biology - Abstract
Aided by extensive spike protein mutation, the SARS-CoV-2 Omicron variant overtook the previously dominant Delta variant. Spike conformation plays an essential role in SARS-CoV-2 evolution via changes in receptor-binding domain (RBD) and neutralizing antibody epitope presentation, affecting virus transmissibility and immune evasion. Here, we determine cryo-EM structures of the Omicron and Delta spikes to understand the conformational impacts of mutations in each. The Omicron spike structure revealed an unusually tightly packed RBD organization with long range impacts that were not observed in the Delta spike. Binding and crystallography revealed increased flexibility at the functionally critical fusion peptide site in the Omicron spike. These results reveal a highly evolved Omicron spike architecture with possible impacts on its high levels of immune evasion and transmissibility.
- Published
- 2022
8. Leveraging Fungal and Human Calcineurin-Inhibitor Structures, Biophysical Data, and Dynamics To Design Selective and Nonimmunosuppressive FK506 Analogs
- Author
-
D. Christopher Cole, William J. Steinbach, Leonard D. Spicer, Benjamin G. Bobay, Praveen R. Juvvadi, Sophie M. C. Gobeil, Ronald A. Venters, and Joseph Heitman
- Subjects
Antifungal Agents ,Calcineurin Pathway ,In silico ,Population ,FK506 ,Calcineurin Inhibitors ,Antifungal drug ,X-ray crystal structures ,Computational biology ,Tacrolimus Binding Protein 1A ,Biology ,Microbiology ,A. fumigatus ,Tacrolimus ,Fungal Proteins ,M. circinelloides ,Virology ,structural biology ,Humans ,Mucormycosis ,Mucor circinelloides ,Amino Acid Sequence ,education ,isothermal titration calorimetry (ITC) ,calcineurin ,education.field_of_study ,Aspergillus fumigatus ,Rational design ,APX879 ,biology.organism_classification ,QR1-502 ,NMR ,molecular dynamics ,Calcineurin ,nuclear magnetic resonance ,FKBP ,Mucor ,Drug Design ,Host-Pathogen Interactions ,FKBP12 ,molecular dynamic (MD) simulations ,Sequence Alignment ,antifungals ,Research Article - Abstract
Calcineurin is a critical enzyme in fungal pathogenesis and antifungal drug tolerance and, therefore, an attractive antifungal target. Current clinically accessible calcineurin inhibitors, such as FK506, are immunosuppressive to humans, so exploiting calcineurin inhibition as an antifungal strategy necessitates fungal specificity in order to avoid inhibiting the human pathway. Harnessing fungal calcineurin-inhibitor crystal structures, we recently developed a less immunosuppressive FK506 analog, APX879, with broad-spectrum antifungal activity and demonstrable efficacy in a murine model of invasive fungal infection. Our overarching goal is to better understand, at a molecular level, the interaction determinants of the human and fungal FK506-binding proteins (FKBP12) required for calcineurin inhibition in order to guide the design of fungus-selective, nonimmunosuppressive FK506 analogs. To this end, we characterized high-resolution structures of the Mucor circinelloides FKBP12 bound to FK506 and of the Aspergillus fumigatus, M. circinelloides, and human FKBP12 proteins bound to the FK506 analog APX879, which exhibits enhanced selectivity for fungal pathogens. Combining structural, genetic, and biophysical methodologies with molecular dynamics simulations, we identify critical variations in these structurally similar FKBP12-ligand complexes. The work presented here, aimed at the rational design of more effective calcineurin inhibitors, indeed suggests that modifications to the APX879 scaffold centered around the C15, C16, C18, C36, and C37 positions provide the potential to significantly enhance fungal selectivity. IMPORTANCE Invasive fungal infections are a leading cause of death in the immunocompromised patient population. The rise in drug resistance to current antifungals highlights the urgent need to develop more efficacious and highly selective agents. Numerous investigations of major fungal pathogens have confirmed the critical role of the calcineurin pathway for fungal virulence, making it an attractive target for antifungal development. Although FK506 inhibits calcineurin, it is immunosuppressive in humans and cannot be used as an antifungal. By combining structural, genetic, biophysical, and in silico methodologies, we pinpoint regions of the FK506 scaffold and a less immunosuppressive analog, APX879, centered around the C15 to C18 and C36 to C37 positions that could be altered with selective extensions and/or deletions to enhance fungal selectivity. This work represents a significant advancement toward realizing calcineurin as a viable target for antifungal drug discovery.
- Published
- 2021
9. 15N, 13C and 1H resonance assignments of FKBP12 proteins from the pathogenic fungi Mucor circinelloides and Aspergillus fumigatus
- Author
-
Benjamin G. Bobay, Leonard D. Spicer, Sophie M. C. Gobeil, and Ronald A. Venters
- Subjects
0303 health sciences ,biology ,Calcineurin ,Aspergillus fumigatus ,FK506 ,030303 biophysics ,Antifungal drug ,biology.organism_classification ,Biochemistry ,Article ,Yeast ,Microbiology ,03 medical and health sciences ,FKBP ,Targeted drug delivery ,Structural Biology ,Mucor circinelloides ,FKBP12 ,Ternary complex ,030304 developmental biology - Abstract
Invasive fungal infections are a leading cause of death in immunocompromised patients and remain difficult to treat since fungal pathogens, like mammals, are eukaryotes and share many orthologous proteins. As a result, current antifungal drugs have limited clinical value, are sometimes toxic, can adversely affect human reaction pathways and are increasingly ineffective due to emerging resistance. One potential antifungal drug, FK506, establishes a ternary complex between the phosphatase, calcineurin, and the 12-kDa peptidyl-prolyl isomerase FK506-binding protein, FKBP12. It has been well established that calcineurin, highly conserved from yeast to mammals, is necessary for invasive fungal disease and is inhibited when in complex with FK506/FKBP12. Unfortunately, FK506 is also immunosuppressive in humans, precluding its usage as an antifungal drug, especially in immunocompromised patients. Whereas the homology between human and fungal calcineurin proteins is > 80%, the human and fungal FKBP12s share 48–58% sequence identity, making them more amenable candidates for drug targeting efforts. Here we report the backbone and sidechain NMR assignments of recombinant FKBP12 proteins from the pathogenic fungi Mucor circinelloides and Aspergillus fumigatus in the apo form and compare these to the backbone assignments of the FK506 bound form. In addition, we report the backbone assignments of the apo and FK506 bound forms of the Homo sapiens FKBP12 protein for evaluation against the fungal forms. These data are the first steps towards defining, at a residue specific level, the impacts of FK506 binding to fungal and mammalian FKBP12 proteins. Our data highlight differences between the human and fungal FKBP12s that could lead to the design of more selective anti-fungal drugs.
- Published
- 2019
10. Effect of natural mutations of SARS-CoV-2 on spike structure, conformation and antigenicity
- Author
-
Victoria Stalls, Kartik Manne, Shana McDowell, Kevin Wiehe, Robert Parks, Megan Kopp, Rory Henderson, Katayoun Mansouri, Katarzyna Janowska, Sophie M. C. Gobeil, Priyamvada Acharya, Bette T. Korber, Dapeng Li, Robert J. Edwards, Kevin O. Saunders, and Barton F. Haynes
- Subjects
Models, Molecular ,Antigenicity ,Protein Conformation ,Allosteric regulation ,Plasma protein binding ,Antibodies, Viral ,Host Adaptation ,medicine.disease_cause ,01 natural sciences ,Article ,03 medical and health sciences ,Protein structure ,Antigen ,biology.animal ,0103 physical sciences ,medicine ,Animals ,Humans ,Protein Interaction Domains and Motifs ,Mink ,Protein Structure, Quaternary ,Receptor ,Antigens, Viral ,Immune Evasion ,030304 developmental biology ,chemistry.chemical_classification ,Genetics ,0303 health sciences ,Mutation ,Multidisciplinary ,010304 chemical physics ,biology ,SARS-CoV-2 ,Chemistry ,Cryoelectron Microscopy ,COVID-19 ,Antibodies, Neutralizing ,Protein Subunits ,Amino Acid Substitution ,Spike Glycoprotein, Coronavirus ,biology.protein ,Angiotensin-Converting Enzyme 2 ,Antibody ,Glycoprotein ,Protein Binding ,Receptors, Coronavirus - Abstract
SummaryNew SARS-CoV-2 variants that have accumulated multiple mutations in the spike (S) glycoprotein enable increased transmission and resistance to neutralizing antibodies. Here, we study the antigenic and structural impacts of the S protein mutations from four variants, one that was involved in transmission between minks and humans, and three that rapidly spread in human populations and originated in the United Kingdom, Brazil or South Africa. All variants either retained or improved binding to the ACE2 receptor. The B.1.1.7 (UK) and B.1.1.28 (Brazil) spike variants showed reduced binding to neutralizing NTD and RBD antibodies, respectively, while the B.1.351 (SA) variant showed reduced binding to both NTD- and RBD-directed antibodies. Cryo-EM structural analyses revealed allosteric effects of the mutations on spike conformations and revealed mechanistic differences that either drive inter-species transmission or promotes viral escape from dominant neutralizing epitopes.HighlightsCryo-EM structures reveal changes in SARS-CoV-2 S protein during inter-species transmission or immune evasion.Adaptation to mink resulted in increased ACE2 binding and spike destabilization.B.1.1.7 S mutations reveal an intricate balance of stabilizing and destabilizing effects that impact receptor and antibody binding.E484K mutation in B.1.351 and B.1.1.28 S proteins drives immune evasion by altering RBD conformation.S protein uses different mechanisms to converge upon similar solutions for altering RBD up/down positioning.
- Published
- 2021
11. SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys
- Author
-
Barton F. Haynes, Esther J. Lee, Mark G. Lewis, Laura L. Sutherland, Robert J. Edwards, Kevin O. Saunders, S. Munir Alam, R. Wes Rountree, Mark A. Tomai, Kartik Manne, Priyamvada Acharya, Anyway B. Kapingidza, C. Todd DeMarco, Sophie M. C. Gobeil, Maggie Barr, Robert A. Seder, Matthew Gagne, Robert Parks, Rachel L. Spreng, Dapeng Li, Gregory D. Sempowski, Aja Sanzone, Hanne Leth Andersen, Ralph S. Baric, Christopher B. Fox, Mihai L. Azoitei, Elizabeth Petzold, Thomas N. Denny, Longping V. Tse, Christopher W. Woods, David C. Montefiori, Drew Weissman, Norbert Pardi, Daniel C. Douek, Haiyan Chen, Katayoun Mansouri, David R. Martinez, Ian N. Moore, Trevor Scobey, Thomas H. Oguin, Kevin Wiehe, Madison Berry, and Fangping Cai
- Subjects
viruses ,fungi ,Biology ,medicine.disease ,Macaque ,Virology ,Article ,Virus ,respiratory tract diseases ,body regions ,Vaccination ,Titer ,Immune system ,Immunization ,biology.animal ,medicine ,biology.protein ,Middle East respiratory syndrome ,Antibody ,skin and connective tissue diseases - Abstract
SUMMARYBetacoronaviruses (betaCoVs) caused the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) outbreaks, and now the SARS-CoV-2 pandemic. Vaccines that elicit protective immune responses against SARS-CoV-2 and betaCoVs circulating in animals have the potential to prevent future betaCoV pandemics. Here, we show that immunization of macaques with a multimeric SARS-CoV-2 receptor binding domain (RBD) nanoparticle adjuvanted with 3M-052-Alum elicited cross-neutralizing antibody responses against SARS-CoV-1, SARS-CoV-2, batCoVs and the UK B.1.1.7 SARS-CoV-2 mutant virus. Nanoparticle vaccination resulted in a SARS-CoV-2 reciprocal geometric mean neutralization titer of 47,216, and robust protection against SARS-CoV-2 in macaque upper and lower respiratory tracts. Importantly, nucleoside-modified mRNA encoding a stabilized transmembrane spike or monomeric RBD protein also induced SARS-CoV-1 and batCoV cross-neutralizing antibodies, albeit at lower titers. These results demonstrate current mRNA vaccines may provide some protection from future zoonotic betaCoV outbreaks, and provide a platform for further development of pan-betaCoV nanoparticle vaccines.
- Published
- 2021
12. The functions of SARS-CoV-2 neutralizing and infection-enhancing antibodies in vitro and in mice and nonhuman primates
- Author
-
Ian N. Moore, Priyamvada Acharya, C. Todd DeMarco, Megan Kopp, Maggie Barr, Andrew N. Macintyre, Sophie M. C. Gobeil, Chuancang Jiang, Ralph S. Baric, Alexandra Schäfer, Derek W. Cain, M. Anthony Moody, Bianca M. Nagata, David R. Martinez, Kevin W. Bock, Robert Parks, Kartik Manne, Laura L. Sutherland, Thomas N. Denny, I-Ting Teng, Victoria Gee-Lai, Giovanna Hernandez, S. Munir Alam, Margaret Deyton, Xiaozhi Lu, John R. Mascola, Dapeng Li, Aaron G. Schmidt, Lautaro G. Perez, Christopher W. Woods, Charlene McDanal, Jared Feldman, Aja Sanzone, Ken Cronin, Barney S. Graham, Katarzyna Janowska, Robert A. Seder, Timothy M. Caradonna, Katayoun Mansouri, Kedamawit Tilahun, Barton F. Haynes, Esther J. Lee, Erica Stover, Elizabeth Petzold, Mahnaz Minai, Tarra Von Holle, Kevin Wiehe, Longping V. Tse, David C. Montefiori, Thomas H. Oguin, Victoria Stalls, Robert J. Edwards, Kevin O. Saunders, Fangping Cai, Trevor Scobey, Blake M. Hauser, Mark G. Lewis, Gregory D. Sempowski, Tongqing Zhou, Andrew Foulger, Peter D. Kwong, and Hanne Leth Andersen
- Subjects
biology ,medicine.diagnostic_test ,business.industry ,viruses ,medicine.medical_treatment ,Fc receptor ,Disease ,Virology ,Article ,In vitro ,Virus ,respiratory tract diseases ,Bronchoalveolar lavage ,Cytokine ,In vivo ,biology.protein ,medicine ,Antibody ,business - Abstract
SummarySARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infectionin vitro, while five non-neutralizing NTD antibodies mediated FcγR-independentin vitroinfection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Nonetheless, three of 31 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, whilein vitroantibody-enhanced infection does not necessarily herald enhanced infectionin vivo, increased lung inflammation can occur in SARS-CoV-2 antibody-infused macaques.
- Published
- 2021
13. Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses
- Author
-
Hanne Leth Andersen, Hana Golding, Matthew Gagne, Robert J. Edwards, Kevin O. Saunders, Barton F. Haynes, Mihai L. Azoitei, Kartik Manne, Gregory D. Sempowski, Dapeng Li, Thomas N. Denny, Esther J. Lee, C. Todd DeMarco, Robert A. Seder, Elizabeth Petzold, Priyamvada Acharya, Surender Khurana, Mahnaz Minai, Mark G. Lewis, Trevor Scobey, R. Wes Rountree, Sophie M. C. Gobeil, Anyway B. Kapingidza, Thomas H. Oguin, Christopher W. Woods, Madison Berry, Rachel L. Spreng, Drew Weissman, Juanjie Tang, Mark A. Tomai, Christopher B. Fox, Ian N. Moore, Laura L. Sutherland, Kevin Wiehe, Ralph S. Baric, Robert Parks, Fangping Cai, Katayoun Mansouri, Norbert Pardi, Kevin W. Bock, Longping V. Tse, Daniel C. Douek, David R. Martinez, David C. Montefiori, Bianca M. Nagata, Aja Sanzone, Maggie Barr, S. Munir Alam, and Haiyan Chen
- Subjects
0301 basic medicine ,Male ,Models, Molecular ,viruses ,Common Cold ,Neutralization ,0302 clinical medicine ,Pandemic ,030212 general & internal medicine ,Neutralizing antibody ,skin and connective tissue diseases ,Vaccines ,Multidisciplinary ,biology ,Vaccination ,virus diseases ,Research Highlight ,Trachea ,Spike Glycoprotein, Coronavirus ,Infectious diseases ,Female ,Antibody ,COVID-19 Vaccines ,Cross Reactions ,Article ,03 medical and health sciences ,Betacoronavirus ,Adjuvants, Immunologic ,medicine ,Animals ,Humans ,Pandemics ,Administration, Intranasal ,SARS-CoV-2 ,fungi ,Outbreak ,COVID-19 ,Viral Vaccines ,medicine.disease ,Virology ,Antibodies, Neutralizing ,respiratory tract diseases ,body regions ,Disease Models, Animal ,030104 developmental biology ,Immunization ,biology.protein ,Middle East respiratory syndrome ,Macaca ,Nanoparticles - Abstract
Betacoronaviruses caused the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome, as well as the current pandemic of SARS coronavirus 2 (SARS-CoV-2)1-4. Vaccines that elicit protective immunity against SARS-CoV-2 and betacoronaviruses that circulate in animals have the potential to prevent future pandemics. Here we show that the immunization of macaques with nanoparticles conjugated with the receptor-binding domain of SARS-CoV-2, and adjuvanted with 3M-052 and alum, elicits cross-neutralizing antibody responses against bat coronaviruses, SARS-CoV and SARS-CoV-2 (including the B.1.1.7, P.1 and B.1.351 variants). Vaccination of macaques with these nanoparticles resulted in a 50% inhibitory reciprocal serum dilution (ID50) neutralization titre of 47,216 (geometric mean) for SARS-CoV-2, as well as in protection against SARS-CoV-2 in the upper and lower respiratory tracts. Nucleoside-modified mRNAs that encode a stabilized transmembrane spike or monomeric receptor-binding domain also induced cross-neutralizing antibody responses against SARS-CoV and bat coronaviruses, albeit at lower titres than achieved with the nanoparticles. These results demonstrate that current mRNA-based vaccines may provide some protection from future outbreaks of zoonotic betacoronaviruses, and provide a multimeric protein platform for the further development of vaccines against multiple (or all) betacoronaviruses.
- Published
- 2021
- Full Text
- View/download PDF
14. D614G mutation alters SARS-CoV-2 spike conformation and enhances protease cleavage at the S1/S2 junction
- Author
-
Priyamvada Acharya, Sophie M. C. Gobeil, Katarzyna Janowska, Megan Kopp, Victoria Stalls, Robert Parks, Katayoun Mansouri, Shana McDowell, Robert J. Edwards, Barton F. Haynes, Kartik Manne, and Rory Henderson
- Subjects
0301 basic medicine ,Mutation rate ,medicine.medical_treatment ,viruses ,Protein domain ,Allosteric regulation ,Molecular Dynamics Simulation ,medicine.disease_cause ,Cleavage (embryo) ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,0302 clinical medicine ,Immunogenicity, Vaccine ,Protein Domains ,medicine ,Humans ,Protein Structure, Quaternary ,skin and connective tissue diseases ,lcsh:QH301-705.5 ,Furin ,Coronavirus ,2P ,allostery ,Protease ,biology ,Chemistry ,Protein Stability ,SARS-CoV-2 ,Cryoelectron Microscopy ,COVID-19 ,spike ,D614G ,Cell biology ,Protein Subunits ,030104 developmental biology ,lcsh:Biology (General) ,Ectodomain ,Mutation ,Proteolysis ,Spike Glycoprotein, Coronavirus ,biology.protein ,030217 neurology & neurosurgery ,Peptide Hydrolases - Abstract
The SARS-CoV-2 spike (S) protein is the target of vaccine design efforts to end the COVID-19 pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic, and are now the dominant form worldwide. Here, we explore spike conformational changes and the effects of the D614G mutation on a soluble S ectodomain construct. Cryo-EM structures reveal altered RBD disposition; antigenicity and proteolysis experiments reveal structural changes and enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage alters the up/down ratio of the Receptor Binding Domains (RBD) in the G614 S ectodomain, demonstrating an allosteric effect on RBD positioning triggered by changes in the SD2 region, that harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 spike conformational landscape and allostery, and have implications for vaccine design., Graphical Abstract, Highlights • SARS-CoV-2 S 2P mutations do not impact its structure, stability or antigenicity. • D614G mutation increases RBD “up” state and enhances S1/S2 junction proteolysis. • Structure and antigenicity reveal allostery between the S1/S2 junction and RBD. • SD2 anchors the mobile RBD and NTD, separating large S1 subunit motions from S2., SARS-CoV-2 spike undergoes large conformational changes during cell fusion. Gobeil et al. identify a subdomain anchor that limits large motions in the receptor binding subunit of the pre-fusion spike from propagating to its fusion subunit. They demonstrate that the D614G mutation increases rate of furin cleavage, which may impact infectivity.
- Published
- 2020
15. D614G mutation alters SARS-CoV-2 spike conformational dynamics and protease cleavage susceptibility at the S1/S2 junction
- Author
-
Victoria Stalls, Katayoun Mansouri, Kartik Manne, Katarzyna Janowska, Barton F. Haynes, Megan Kopp, Sophie M. C. Gobeil, Rory Henderson, Priyamvada Acharya, Robert J. Edwards, Shana McDowell, and Robert Parks
- Subjects
Antigenicity ,Mutation rate ,Protease ,medicine.diagnostic_test ,biology ,Chemistry ,medicine.medical_treatment ,Proteolysis ,viruses ,Allosteric regulation ,Cleavage (embryo) ,Article ,Cell biology ,Ectodomain ,medicine ,biology.protein ,Furin - Abstract
The SARS-CoV-2 spike (S) protein is the target of vaccine design efforts to end the COVID-19 pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic, and are now the dominant form worldwide. Here, we analyze the D614G mutation in the context of a soluble S ectodomain construct. Cryo-EM structures, antigenicity and proteolysis experiments suggest altered conformational dynamics resulting in enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage altered the conformational dynamics of the Receptor Binding Domains (RBD) in the G614 S ectodomain, demonstrating an allosteric effect on the RBD dynamics triggered by changes in the SD2 region, that harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 spike conformational dynamics and allostery, and have implications for vaccine design.HighlightsSARS-CoV-2 S ectodomains with or without the K986P, V987P mutations have similar structures, antigenicity and stability.The D614G mutation alters S protein conformational dynamics.D614G enhances protease cleavage susceptibility at the S protein furin cleavage site.Cryo-EM structures reveal allosteric effect of changes at the S1/S2 junction on RBD dynamics.
- Published
- 2020
16. Controlling the SARS-CoV-2 spike glycoprotein conformation
- Author
-
Rory Henderson, Barton F. Haynes, Megan Kopp, Priyamvada Acharya, Sophie M. C. Gobeil, Allen L. Hsu, Mario J. Borgnia, Robert J. Edwards, Dapeng Li, Rob Parks, Victoria Stalls, Katarzyna Janowska, and Katayoun Mansouri
- Subjects
Models, Molecular ,Viral protein ,Protein Conformation ,Computer science ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,viruses ,Protein subunit ,Protein domain ,Virus Neutralization ,Computational biology ,Biology ,medicine.disease_cause ,Article ,03 medical and health sciences ,0302 clinical medicine ,Protein structure ,Protein Domains ,Structural Biology ,medicine ,Binding site ,Molecular Biology ,030304 developmental biology ,Coronavirus ,chemistry.chemical_classification ,0303 health sciences ,Binding Sites ,Cell fusion ,Cryoelectron Microscopy ,Rational design ,Microscopy, Electron ,Protein Subunits ,chemistry ,Ectodomain ,Mutation ,Spike Glycoprotein, Coronavirus ,Spike (software development) ,Glycoprotein ,030217 neurology & neurosurgery - Abstract
The coronavirus (CoV) viral host cell fusion spike (S) protein is the primary immunogenic target for virus neutralization and the current focus of many vaccine design efforts. The highly flexible S-protein, with its mobile domains, presents a moving target to the immune system. Here, to better understand S-protein mobility, we implemented a structure-based vector analysis of available β-CoV S-protein structures. We found that despite overall similarity in domain organization, different β-CoV strains display distinct S-protein configurations. Based on this analysis, we developed two soluble ectodomain constructs in which the highly immunogenic and mobile receptor binding domain (RBD) is locked in either the all-RBDs ‘down’ position or is induced to display a previously unobserved in SARS-CoV-2 2-RBDs ‘up’ configuration. These results demonstrate that the conformation of the S-protein can be controlled via rational design and provide a framework for the development of engineered coronavirus spike proteins for vaccine applications.
- Published
- 2020
- Full Text
- View/download PDF
17. Leveraging Fungal Calcineurin-Inhibitor Structures, Biophysics and Dynamics to Design Selective and Non-Immunosuppressive FK506 Analogs
- Author
-
William J. Steinbach, Sophie M. C. Gobeil, D. Christopher Cole, Leonard D. Spicer, Praveen R. Juvvadi, Benjamin G. Bobay, Ronald A. Venters, and Joseph Heitman
- Subjects
0303 health sciences ,education.field_of_study ,010304 chemical physics ,Calcineurin Pathway ,In silico ,Population ,Antifungal drug ,Rational design ,Drug resistance ,Computational biology ,Biology ,01 natural sciences ,3. Good health ,Calcineurin ,03 medical and health sciences ,FKBP ,0103 physical sciences ,education ,030304 developmental biology - Abstract
Calcineurin is a critical enzyme in fungal pathogenesis and antifungal drug tolerance and, therefore, an attractive antifungal target. Current clinically-accessible calcineurin inhibitors, such as FK506, are immunosuppressive to humans, so exploiting calcineurin inhibition as an antifungal strategy necessitates fungal-specificity in order to avoid inhibiting the human pathway. Harnessing fungal calcineurin-inhibitor crystal structures, we recently developed a less immunosuppressive FK506 analog, APX879, with broad-spectrum antifungal activity and demonstrable efficacy in a murine model of invasive fungal infection. Our overarching goal is to better understand, at a molecular level, the interaction determinants of the human and fungal FK506-binding proteins (FKBP12) required for calcineurin inhibition in order to guide the design of fungal-selective, non-immunosuppressive FK506 analogs. To this end, we characterized high-resolution structures of theM. circinelloidesFKBP12 bound to FK506, and of theA. fumigatus, M. circinelloidesand human FKBP12 proteins bound to the FK506 analog, APX879, which exhibits enhanced selectivity for fungal pathogens. Combining structural, genetic and biophysical methodologies with molecular dynamics simulations, we identify critical variations in these structurally similar FKBP12-ligand complexes that will guide the rational design of inhibitors with enhanced fungal-selectivity.Significance statementInvasive fungal infections are a leading cause of death in the immunocompromised patient population. The rise in drug resistance to current antifungals highlights the urgent need to develop more efficacious and highly selective agents. Numerous investigations of major fungal pathogens have confirmed the critical role of the calcineurin pathway for fungal virulence, making it an attractive target for antifungal development. Although FK506 inhibits calcineurin, it is immunosuppressive in humans and cannot be used as an antifungal. By combining structural, genetic, biophysical, andin silicomethodologies, we pinpoint regions of FK506 and a less immunosuppressive analog, APX879, that could be altered to enhance fungal selectivity. This work represents a significant advancement toward realizing calcineurin as a viable target for antifungal drug discovery.
- Published
- 2020
18. In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies
- Author
-
Mark G. Lewis, Andrew N. Macintyre, Robert Parks, Fangping Cai, S. Munir Alam, Dapeng Li, Trevor Scobey, Alexandra Schäfer, Timothy M. Caradonna, C. Todd DeMarco, Kevin W. Bock, Ian N. Moore, Kedamawit Tilahun, Charlene McDanal, Thomas H. Oguin, I-Ting Teng, Priyamvada Acharya, Andrew Foulger, Tarra Von Holle, Elizabeth Petzold, Longping V. Tse, Christopher W. Woods, Megan Kopp, Robert J. Edwards, Bianca M. Nagata, Mahnaz Minai, M. Anthony Moody, Thomas N. Denny, Victoria Gee-Lai, John R. Mascola, Sophie M. C. Gobeil, Kevin Wiehe, Lautaro G. Perez, Tongqing Zhou, Chuancang Jiang, Kevin O. Saunders, David C. Montefiori, Aja Sanzone, Erica Stover, Katarzyna Janowska, Kartik Manne, Gregory D. Sempowski, Peter D. Kwong, Barton F. Haynes, Blake M. Hauser, Wes Rountree, Derek W. Cain, David R. Martinez, Barney S. Graham, Maggie Barr, Esther J. Lee, Ralph S. Baric, Kenneth D. Cronin, Margaret Deyton, Victoria Stalls, Xiaozhi Lu, Jared Feldman, Katayoun Mansouri, Hanne Leth Andersen, Laura L. Sutherland, Giovanna Hernandez, Robert A. Seder, Yunfei Wang, and Aaron G. Schmidt
- Subjects
Male ,N-terminal domain ,viruses ,Fc receptor ,Inflammation ,Antibodies, Viral ,Virus Replication ,Article ,General Biochemistry, Genetics and Molecular Biology ,Virus ,infection enhancement ,Proinflammatory cytokine ,Mice ,Protein Domains ,In vivo ,medicine ,Animals ,Humans ,antibody-dependent enhancement ,skin and connective tissue diseases ,Lung ,Mice, Inbred BALB C ,biology ,medicine.diagnostic_test ,SARS-CoV-2 ,Receptors, IgG ,cross-neutralization ,fungi ,electron micrograph ,COVID-19 ,neutralizing antibody ,Haplorhini ,Viral Load ,respiratory system ,Antibodies, Neutralizing ,In vitro ,respiratory tract diseases ,Bronchoalveolar lavage ,in vivo protection ,Spike Glycoprotein, Coronavirus ,Immunology ,biology.protein ,Cytokines ,Female ,receptor-binding domain ,Antibody ,medicine.symptom ,Bronchoalveolar Lavage Fluid ,RNA, Guide, Kinetoplastida - Abstract
SARS-CoV-2-neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) or the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques., Graphical abstract, Convalescent human-derived SARS-CoV-2 RBD and NTD antibodies mediated neutralization as well as infection enhancement in vitro, yet infusion of these antibodies in mice or cynomolgus macaques resulted in suppression of virus replication.
- Published
- 2021
19. Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies
- Author
-
Katarzyna Janowska, Guillaume Stewart-Jones, Ye Zhou, Bhavna Hora, Naomi Bronkema, Tyler Evangelous, Alberto Bartesaghi, John R. Perfect, Hui Li, Robert A. Seder, Michael S. Seaman, Celia C. LaBranche, A. Yousef Abuahmad, Jordan Sprenz, Joseph R. Francica, M. Anthony Moody, Baptiste Aussedat, S. Munir Alam, Garnett Kelsoe, Sampa Santra, Barton F. Haynes, Richard Laga, Priyamvada Acharya, Katayoun Mansouri, Daniela Fera, Victoria Stalls, Nathan I. Nicely, Margaret Deyton, Allen L. Hsu, Madison Berry, George M. Shaw, Megan Kopp, William E. Walkowicz, David C. Montefiori, Gregory D. Sempowski, Matthew S. Lee, Sophie M. C. Gobeil, Mario J. Borgnia, Todd Bradley, Thomas H. Oguin, R. Ryan Meyerhoff, Robert Parks, Kevin Wiehe, Mattia Bonsignori, Peter D. Kwong, Kartik Manne, Geoffrey M. Lynn, Rory Henderson, Robert J. Edwards, Wilton B. Williams, Andrew Foulger, and Kevin O. Saunders
- Subjects
Glycosylation ,HIV Infections ,marginal zone B cells ,HIV Antibodies ,medicine.disease_cause ,Immunoglobulin D ,Epitope ,chemistry.chemical_compound ,Epitopes ,0302 clinical medicine ,FDG Abs ,glycan-dependent Ab binding ,0303 health sciences ,B-Lymphocytes ,Vaccines ,biology ,Immunoglobulin Fab Fragments ,env Gene Products, Human Immunodeficiency Virus ,virus diseases ,Spike Glycoprotein, Coronavirus ,Simian Immunodeficiency Virus ,natural Abs ,Antibody ,Dimerization ,Glycan ,Receptors, Antigen, B-Cell ,SARS-CoV-2 spike glycans ,IgM-memory B cells ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Antigen ,Polysaccharides ,medicine ,Animals ,Humans ,030304 developmental biology ,SARS-CoV-2 ,HIV-1 Env glycans ,COVID-19 ,Simian immunodeficiency virus ,Virology ,Antibodies, Neutralizing ,Macaca mulatta ,carbohydrates (lipids) ,chemistry ,biology.protein ,HIV-1 ,Fab dimerization ,030217 neurology & neurosurgery ,Broadly Neutralizing Antibodies - Abstract
Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells., Graphical abstract, Structural and functional analyses identify a category of glycan-reactive antibodies in macaques and humans that are marked by the dimerization of the antigen-binding fragment. These antibodies are involved in HIV neutralization and also recognize the S2 protein of SARS-CoV-2.
- Published
- 2021
20. Harnessing calcineurin-FK506-FKBP12 crystal structures from invasive fungal pathogens to develop antifungal agents
- Author
-
Sophie M. C. Gobeil, Leonard D. Spicer, David A. Fox, Jackie J. Lin, Maria Ciofani, William J. Steinbach, Michael J. Hoy, Praveen R. Juvvadi, Ronald A. Venters, Blake C. Barrington, Zanetta Chang, Maria A. Schumacher, Anna F. Averette, Joseph Heitman, Michael Trzoss, Mitchell Mutz, Ying-Lien Chen, Joshua D. Wheaton, Xiaoming Li, Soo Chan Lee, Benjamin G. Bobay, and D. Christopher Cole
- Subjects
Male ,0301 basic medicine ,Antifungal Agents ,General Physics and Astronomy ,Tacrolimus Binding Protein 1A ,Crystallography, X-Ray ,Aspergillus fumigatus ,Mice ,Candida albicans ,Drug Discovery ,polycyclic compounds ,lcsh:Science ,Cells, Cultured ,Multidisciplinary ,biology ,Chemistry ,Calcineurin ,Cryptococcosis ,3. Good health ,FKBP ,cardiovascular system ,Female ,Structure-based drug design ,Mice, Inbred A ,Coccidioides immitis ,Science ,Calcineurin Inhibitors ,030106 microbiology ,Virulence ,Molecular Dynamics Simulation ,Tacrolimus ,Article ,General Biochemistry, Genetics and Molecular Biology ,Microbiology ,03 medical and health sciences ,Hydrolase ,Animals ,Aspergillosis ,X-ray crystallography ,Cryptococcus neoformans ,Lead optimization ,Binding Sites ,Coccidioides ,organic chemicals ,General Chemistry ,biology.organism_classification ,Mice, Inbred C57BL ,enzymes and coenzymes (carbohydrates) ,030104 developmental biology ,lcsh:Q - Abstract
Calcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection., FK506 is a potential antifungal compound that inhibits calcineurin, but it also has immunosuppressive activity. Here, Juvvadi et al. report the structure of FK506 in complex with the FK506-binding protein FKPB12 and calcineurin, and design a less immunosuppresive FK506 analog with antifungal activity in mice.
- Published
- 2019
21. The Structural Dynamics of Engineered β-Lactamases Vary Broadly on Three Timescales yet Sustain Native Function
- Author
-
Donald Gagné, Jürgen Pleiss, Maximillian C C J C Ebert, Albert M. Berghuis, Jaeok Park, Joelle N. Pelletier, Sophie M. C. Gobeil, Nicolas Doucet, PROTEO, The Quebec Network for Research on Protein Function, Engineering, and Applications, Institut Armand Frappier (INRS-IAF), Réseau International des Instituts Pasteur (RIIP)-Institut National de la Recherche Scientifique [Québec] (INRS)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Recherche Scientifique [Québec] (INRS)-Université de Sherbrooke (UdeS)-Université Laval [Québec] (ULaval)-McGill University = Université McGill [Montréal, Canada]-University of Ottawa [Ottawa]-Université du Québec à Trois-Rivières (UQTR)-Université de Montréal (UdeM)-TransBiotech, Lévis-Concordia University [Montreal]-Université du Québec à Montréal = University of Québec in Montréal (UQAM), Département de chimie [UdeM-Montréal], Université de Montréal (UdeM), Groupe de Recherche Axé sur la Structure des Protéines [Montréal] (GRASP), McGill University = Université McGill [Montréal, Canada], Department of Biochemistry [Montréal], Réseau International des Instituts Pasteur (RIIP)-Institut National de la Recherche Scientifique [Québec] (INRS), University of Stuttgart, This work was supported by NSERC Discovery Grants 227853 (J.N.P) and 2016-07555 (N.D.), and NIH award R01GM105978 (N.D.). S.G. is a FRQ-NT Graduate Scholar, M.E. a NSERC Vanier Canada Graduate Scholar and D.G. an NSERC Alexander Graham Bell Canada Graduate Scholar. N.D. holds a FRQ-S Research Scholar Junior 2 Career Award., The authors thank M. Meyer and F. Arnold for the original cTEM-19m construct, and T. Sprules and S. Al-Abdul-Wahid (Québec/Eastern Canada High Field NMR Facility) and M. Osborne (IRIC Biophysics Research Facilities). Computational resources were provided by Calcul Québec, Compute Canada, and High Performance Computing Center Stuttgart.
- Subjects
Models, Molecular ,0301 basic medicine ,Protein Conformation ,[SDV]Life Sciences [q-bio] ,In silico ,lcsh:Medicine ,Computational biology ,Crystallography, X-Ray ,Protein Engineering ,Article ,beta-Lactamases ,03 medical and health sciences ,0302 clinical medicine ,Enzyme system ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,lcsh:Science ,Nuclear Magnetic Resonance, Biomolecular ,X-ray crystallography ,Protein function ,Binding Sites ,Multidisciplinary ,Chemistry ,β lactamases ,Protein dynamics ,lcsh:R ,Dynamics (mechanics) ,Protein engineering ,Kinetics ,030104 developmental biology ,lcsh:Q ,Protein design ,Solution-state NMR ,030217 neurology & neurosurgery ,Function (biology) ,Protein Binding - Abstract
Understanding the principles of protein dynamics will help guide engineering of protein function: altering protein motions may be a barrier to success or may be an enabling tool for protein engineering. The impact of dynamics on protein function is typically reported over a fraction of the full scope of motional timescales. If motional patterns vary significantly at different timescales, then only by monitoring motions broadly will we understand the impact of protein dynamics on engineering functional proteins. Using an integrative approach combining experimental and in silico methodologies, we elucidate protein dynamics over the entire span of fast to slow timescales (ps to ms) for a laboratory-engineered system composed of five interrelated β-lactamases: two natural homologs and three laboratory-recombined variants. Fast (ps-ns) and intermediate (ns-µs) dynamics were mostly conserved. However, slow motions (µs-ms) were few and conserved in the natural homologs yet were numerous and widely dispersed in their recombinants. Nonetheless, modified slow dynamics were functionally tolerated. Crystallographic B-factors from high-resolution X-ray structures were partly predictive of the conserved motions but not of the new slow motions captured in our solution studies. Our inspection of protein dynamics over a continuous range of timescales vividly illustrates the complexity of dynamic impacts of protein engineering as well as the functional tolerance of an engineered enzyme system to new slow motions.
- Published
- 2019
22. Development of sulfahydantoin derivatives as β-lactamase inhibitors
- Author
-
Laurie Bédard, Lorea Alejaldre, Joelle N. Pelletier, Camille Lapointe Verreault, Charles-Olivier Normandeau, Claudèle Lemay-St-Denis, Rosalie Lamoureux, Sophie M. C. Gobeil, Normand Voyer, and Pierre-Alexandre Paquet-Côté
- Subjects
Clinical Biochemistry ,Pharmaceutical Science ,01 natural sciences ,Biochemistry ,beta-Lactamases ,Structure-Activity Relationship ,chemistry.chemical_compound ,Drug Development ,β lactamase inhibitor ,Drug Discovery ,Ic50 values ,Humans ,Molecule ,Molecular Biology ,chemistry.chemical_classification ,Chlorosulfonyl isocyanate ,Dose-Response Relationship, Drug ,Molecular Structure ,Sulfur Compounds ,010405 organic chemistry ,Organic Chemistry ,Combinatorial chemistry ,0104 chemical sciences ,3. Good health ,010404 medicinal & biomolecular chemistry ,Enzyme ,chemistry ,Molecular Medicine ,beta-Lactamase Inhibitors - Abstract
Sulfahydantoin-based molecules may provide a means to counteract antibiotic resistance, which is on the rise. These molecules may act as inhibitors of β-lactamase enzymes, which are key in some resistance mechanisms. In this paper, we report on the synthesis of 6 novel sulfahydantoin derivatives by the key reaction of chlorosulfonyl isocyanate to form α-amino acid derived sulfamides, and their cyclization into sulfahydantoins. The synthesis is rapid and provides the target compounds in 8 steps. We investigated their potential as β-lactamase inhibitors using two common Class A β-lactamases, TEM-1 and the prevalent extended-spectrum TEM-15. Two compounds, 3 and 6, show substantial inhibition of the β-lactamases with IC50 values between 130 and 510 μM and inferred Ki values between 32 and 55 μM.
- Published
- 2021
23. 15N, 13C and 1H backbone resonance assignments of an artificially engineered TEM-1/PSE-4 class A β-lactamase chimera and its deconvoluted mutant
- Author
-
Nicolas Doucet, Donald Gagné, Joelle N. Pelletier, Sophie M. C. Gobeil, Université de Montréal (UdeM), Université Laval [Québec] (ULaval), The Québec Network for Research on Protein Function, Engineering and Applications, Institut Armand Frappier (INRS-IAF), Institut National de la Recherche Scientifique [Québec] (INRS)-Réseau International des Instituts Pasteur (RIIP), CUNY Graduate Center (The Graduate Center), City University of New York [New York] (CUNY), McGill University = Université McGill [Montréal, Canada], and This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants RGPIN 227853 and 402623 (to J.N.P and N.D, respectively), in addition to a grant from the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under Award No. R01GM105978 (to N.D.). S.G. is the recipient of a FRQNT Graduate Scholarship and D.G. is the recipient of an NSERC Alexander Graham Bell Canada Graduate Scholarship. N.D. holds a Fonds de Recherche Québec – Santé (FRQS) Research Scholar Junior 1 Career Award.
- Subjects
0301 basic medicine ,Stereochemistry ,Antibiotic resistance ,[SDV]Life Sciences [q-bio] ,Mutant ,Morin ,Biology ,Tritium ,Biochemistry ,Article ,Protein Structure, Secondary ,beta-Lactamases ,law.invention ,03 medical and health sciences ,Chimera (genetics) ,chemistry.chemical_compound ,Structural Biology ,law ,TEM-1 ,PSE-4 ,Nuclear Magnetic Resonance, Biomolecular ,β-Lactamase ,Genetics ,chemistry.chemical_classification ,Carbon Isotopes ,Nitrogen Isotopes ,Functional protein ,Chimera ,Protein engineering ,Fusion protein ,Recombinant Proteins ,Amino acid ,030104 developmental biology ,chemistry ,Recombinant DNA ,Mutant Proteins - Abstract
The widespread use of β-lactam antibiotics has given rise to a dramatic increase in clinically-relevant β-lactamases. Understanding the structure/function relation in these variants is essential to better address the ever-growing incidence of antibiotic resistance. We previously reported the backbone resonance assignments of a chimeric protein constituted of segments of the class A β-lactamases TEM-1 and PSE-4 (Morin et al. in Biomol NMR Assign 4:127–130, 2010. doi: 10.1007/s12104-010-9227-8 ). That chimera, cTEM17m, held 17 amino acid substitutions relative to TEM-1 β-lactamase, resulting in a well-folded and fully functional protein with increased dynamics. Here we report the 1H, 13C and 15N backbone resonance assignments of chimera cTEM-19m, which includes 19 substitutions and exhibits increased active-site perturbation, as well as one of its deconvoluted variants, as the first step in the analysis of their dynamic behaviours.
- Published
- 2016
24. Maintenance of native-like protein dynamics may not be required for engineering functional proteins
- Author
-
Nicolas Doucet, Sophie M. C. Gobeil, Joelle N. Pelletier, Jaeok Park, Donald Gagné, Christopher M. Clouthier, Albert M. Berghuis, Université Laval [Québec] (ULaval), Université de Montréal (UdeM), McGill University = Université McGill [Montréal, Canada], Institut Armand Frappier (INRS-IAF), Institut National de la Recherche Scientifique [Québec] (INRS)-Réseau International des Instituts Pasteur (RIIP), Center for green chemistry and catalysis (CCVC), and This work was supported by National Sciences and Engineering Research Council of Canada Discovery Grants Program—Individual #227853 (to J.N.P.) and #402623 (to N.D.) and by Canadian Institutes of Health Research Grant MOP-13107 (to A.M.B.).
- Subjects
[SDV.BIO]Life Sciences [q-bio]/Biotechnology ,General interest ,Recombinant Fusion Proteins ,[SDV]Life Sciences [q-bio] ,Clinical Biochemistry ,Chimeric enzyme ,MESH: Catalytic Domain ,MESH: Protein Structure, Secondary ,MESH: beta-Lactamases ,Computational biology ,Biology ,Molecular Dynamics Simulation ,Crystallography, X-Ray ,Protein Engineering ,Biochemistry ,Protein Structure, Secondary ,beta-Lactamases ,Conserved sequence ,Chimera (genetics) ,[SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases ,Catalytic Domain ,Drug Discovery ,Hydrolase ,MESH: Nuclear Magnetic Resonance, Biomolecular ,MESH: Recombinant Fusion Proteins ,[CHIM]Chemical Sciences ,MESH: Molecular Dynamics Simulation ,MESH: Proteins ,Molecular Biology ,Nuclear Magnetic Resonance, Biomolecular ,Pharmacology ,Genetics ,Protein function ,Nitrogen Isotopes ,Protein dynamics ,Proteins ,General Medicine ,MESH: Crystallography, X-Ray ,MESH: Protein Engineering ,Molecular Medicine ,Recombination ,MESH: Nitrogen Isotopes - Abstract
International audience; Proteins are dynamic systems, and understanding dynamics is critical for fully understanding protein function. Therefore, the question of whether laboratory engineering has an impact on protein dynamics is of general interest. Here, we demonstrate that two homologous, naturally evolved enzymes with high degrees of structural and functional conservation also exhibit conserved dynamics. Their similar set of slow timescale dynamics is highly restricted, consistent with evolutionary conservation of a functionally important feature. However, we also show that dynamics of a laboratory-engineered chimeric enzyme obtained by recombination of the two homologs exhibits striking difference on the millisecond timescale, despite function and high-resolution crystal structure (1.05 Å) being conserved. The laboratory-engineered chimera is thus functionally tolerant to modified dynamics on the timescale of catalytic turnover. Tolerance to dynamic variation implies that maintenance of native-like protein dynamics may not be required when engineering functional proteins.
- Published
- 2014
25. Chimeric β-lactamases: global conservation of parental function and fast time-scale dynamics with increased slow motions
- Author
-
Joelle N. Pelletier, Nicolas Doucet, Sébastien Morin, Jonathan Blanchet, Elisabeth Bon Nguyen, Sophie M. C. Gobeil, Stéphane M. Gagné, Christopher M. Clouthier, PROTEO, The Quebec Network for Research on Protein Function, Engineering, and Applications, Institut Armand Frappier (INRS-IAF), Réseau International des Instituts Pasteur (RIIP)-Institut National de la Recherche Scientifique [Québec] (INRS)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Recherche Scientifique [Québec] (INRS)-Université de Sherbrooke (UdeS)-Université Laval [Québec] (ULaval)-McGill University = Université McGill [Montréal, Canada]-University of Ottawa [Ottawa]-Université du Québec à Trois-Rivières (UQTR)-Université de Montréal (UdeM)-TransBiotech, Lévis-Concordia University [Montreal]-Université du Québec à Montréal = University of Québec in Montréal (UQAM), Département de chimie [UdeM-Montréal], Université de Montréal (UdeM), Département de Biochimie, Microbiologie et Bioinformatique, Université Laval [Québec] (ULaval), Département de Biochimie, Réseau International des Instituts Pasteur (RIIP)-Institut National de la Recherche Scientifique [Québec] (INRS), Département de Biochimie et de Microbiologie, CREFSIP, and This work was supported by operating grants from the Fonds de la recherche en santé du Québec (FRSQ), PROTEO-Fonds québécois de la recherche sur la nature et les technologies (FQRNT) and the Natural Sciences and Engineering Research Council of Canada (NSERC), and infrastructure grants from the Canada Foundation for Innovation (Innovation and New Opportunity). S. Morin is the recipient of scholarships from NSERC and FRSQ. S. Gobeil is the recipient of scholarships from PROTEO and FQRNT
- Subjects
Circular dichroism ,Magnetic Resonance Spectroscopy ,MESH: Protein Structure, Secondary ,MESH: beta-Lactamases ,Molecular Dynamics ,Biochemistry ,01 natural sciences ,Protein Structure, Secondary ,law.invention ,MESH: Circular Dichroism ,Computational Chemistry ,law ,[SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases ,Macromolecular Structure Analysis ,Fluorometry ,Biomacromolecule-Ligand Interactions ,Genetics ,0303 health sciences ,Multidisciplinary ,MESH: Kinetics ,Chemistry ,Circular Dichroism ,Physics ,Applied Chemistry ,Nuclear magnetic resonance spectroscopy ,Recombinant Proteins ,Enzymes ,Recombinant DNA ,Medicine ,Synthetic Biology ,Recombination ,Research Article ,Protein Structure ,Nuclear Magnetic Resonance ,Science ,Kinetics ,010402 general chemistry ,beta-Lactamases ,03 medical and health sciences ,Chimera (genetics) ,Biology ,030304 developmental biology ,Enzyme Kinetics ,MESH: Magnetic Resonance Spectroscopy ,MESH: Fluorometry ,Proteins ,Computational Biology ,Protein engineering ,0104 chemical sciences ,Chemical Properties ,Enzyme Structure ,Biophysics ,Homologous recombination - Abstract
International audience; Enzyme engineering has been facilitated by recombination of close homologues, followed by functional screening. In one such effort, chimeras of two class-A β-lactamases - TEM-1 and PSE-4 - were created according to structure-guided protein recombination and selected for their capacity to promote bacterial proliferation in the presence of ampicillin (Voigt et al., Nat. Struct. Biol. 2002 9:553). To provide a more detailed assessment of the effects of protein recombination on the structure and function of the resulting chimeric enzymes, we characterized a series of functional TEM-1/PSE-4 chimeras possessing between 17 and 92 substitutions relative to TEM-1 β-lactamase. Circular dichroism and thermal scanning fluorimetry revealed that the chimeras were generally well folded. Despite harbouring important sequence variation relative to either of the two 'parental' β-lactamases, the chimeric β-lactamases displayed substrate recognition spectra and reactivity similar to their most closely-related parent. To gain further insight into the changes induced by chimerization, the chimera with 17 substitutions was investigated by NMR spin relaxation. While high order was conserved on the ps-ns timescale, a hallmark of class A β-lactamases, evidence of additional slow motions on the µs-ms timescale was extracted from model-free calculations. This is consistent with the greater number of resonances that could not be assigned in this chimera relative to the parental β-lactamases, and is consistent with this well-folded and functional chimeric β-lactamase displaying increased slow time-scale motions.
- Published
- 2012
26. Backbone resonance assignments of an artificially engineered TEM-1/PSE-4 Class A β-lactamase chimera
- Author
-
Sébastien Morin, Sophie M. C. Gobeil, Stéphane M. Gagné, Christopher M. Clouthier, and Joelle N. Pelletier
- Subjects
Chromogenic ,Stereochemistry ,Functional features ,Kinetics ,Protein engineering ,Biology ,Protein Engineering ,Biochemistry ,Recombinant Proteins ,beta-Lactamases ,law.invention ,Chimera (genetics) ,Structural Biology ,law ,Recombinant DNA ,Enzyme kinetics ,Sequence variation ,Nuclear Magnetic Resonance, Biomolecular - Abstract
The rapid evolution of Class A β-lactamases, which procure resistance to an increasingly broad panel of β-lactam antibiotics, underscores the urgency to better understand the relation between their sequence variation and their structural and functional features. To date, more than 300 clinically-relevant β-lactamase variants have been reported, and this number continues to increase. With the aim of obtaining insights into the evolutionary potential of β-lactamases, an artificially engineered, catalytically active chimera of the Class A TEM-1 and PSE-4 β-lactamases is under study by kinetics and NMR. Here we report the (1)H, (13)C and (15)N backbone resonance assignments for the 30 kDa chimera cTEM-17m. Despite its high molecular weight, the data provide evidence that this artificially-evolved chimeric enzyme is well folded. The hydrolytic activity of cTEM-17m was determined using the chromogenic substrate CENTA, with K (M) = 160 ± 35 μM and k (cat) = 20 ± 4 s(-1), which is in the same range as the values for TEM-1 and PSE-4 β-lactamases.
- Published
- 2009
27. Leveraging Fungal and Human Calcineurin-Inhibitor Structures, Biophysical Data, and Dynamics To Design Selective and Nonimmunosuppressive FK506 Analogs
- Author
-
Sophie M.-C. Gobeil, Benjamin G. Bobay, Praveen R. Juvvadi, D. Christopher Cole, Joseph Heitman, William J. Steinbach, Ronald A. Venters, and Leonard D. Spicer
- Subjects
FK506 ,FKBP12 ,APX879 ,antifungals ,M. circinelloides ,A. fumigatus ,Microbiology ,QR1-502 - Abstract
ABSTRACT Calcineurin is a critical enzyme in fungal pathogenesis and antifungal drug tolerance and, therefore, an attractive antifungal target. Current clinically accessible calcineurin inhibitors, such as FK506, are immunosuppressive to humans, so exploiting calcineurin inhibition as an antifungal strategy necessitates fungal specificity in order to avoid inhibiting the human pathway. Harnessing fungal calcineurin-inhibitor crystal structures, we recently developed a less immunosuppressive FK506 analog, APX879, with broad-spectrum antifungal activity and demonstrable efficacy in a murine model of invasive fungal infection. Our overarching goal is to better understand, at a molecular level, the interaction determinants of the human and fungal FK506-binding proteins (FKBP12) required for calcineurin inhibition in order to guide the design of fungus-selective, nonimmunosuppressive FK506 analogs. To this end, we characterized high-resolution structures of the Mucor circinelloides FKBP12 bound to FK506 and of the Aspergillus fumigatus, M. circinelloides, and human FKBP12 proteins bound to the FK506 analog APX879, which exhibits enhanced selectivity for fungal pathogens. Combining structural, genetic, and biophysical methodologies with molecular dynamics simulations, we identify critical variations in these structurally similar FKBP12-ligand complexes. The work presented here, aimed at the rational design of more effective calcineurin inhibitors, indeed suggests that modifications to the APX879 scaffold centered around the C15, C16, C18, C36, and C37 positions provide the potential to significantly enhance fungal selectivity. IMPORTANCE Invasive fungal infections are a leading cause of death in the immunocompromised patient population. The rise in drug resistance to current antifungals highlights the urgent need to develop more efficacious and highly selective agents. Numerous investigations of major fungal pathogens have confirmed the critical role of the calcineurin pathway for fungal virulence, making it an attractive target for antifungal development. Although FK506 inhibits calcineurin, it is immunosuppressive in humans and cannot be used as an antifungal. By combining structural, genetic, biophysical, and in silico methodologies, we pinpoint regions of the FK506 scaffold and a less immunosuppressive analog, APX879, centered around the C15 to C18 and C36 to C37 positions that could be altered with selective extensions and/or deletions to enhance fungal selectivity. This work represents a significant advancement toward realizing calcineurin as a viable target for antifungal drug discovery.
- Published
- 2021
- Full Text
- View/download PDF
28. Chimeric β-lactamases: global conservation of parental function and fast time-scale dynamics with increased slow motions.
- Author
-
Christopher M Clouthier, Sébastien Morin, Sophie M C Gobeil, Nicolas Doucet, Jonathan Blanchet, Elisabeth Nguyen, Stéphane M Gagné, and Joelle N Pelletier
- Subjects
Medicine ,Science - Abstract
Enzyme engineering has been facilitated by recombination of close homologues, followed by functional screening. In one such effort, chimeras of two class-A β-lactamases - TEM-1 and PSE-4 - were created according to structure-guided protein recombination and selected for their capacity to promote bacterial proliferation in the presence of ampicillin (Voigt et al., Nat. Struct. Biol. 2002 9:553). To provide a more detailed assessment of the effects of protein recombination on the structure and function of the resulting chimeric enzymes, we characterized a series of functional TEM-1/PSE-4 chimeras possessing between 17 and 92 substitutions relative to TEM-1 β-lactamase. Circular dichroism and thermal scanning fluorimetry revealed that the chimeras were generally well folded. Despite harbouring important sequence variation relative to either of the two 'parental' β-lactamases, the chimeric β-lactamases displayed substrate recognition spectra and reactivity similar to their most closely-related parent. To gain further insight into the changes induced by chimerization, the chimera with 17 substitutions was investigated by NMR spin relaxation. While high order was conserved on the ps-ns timescale, a hallmark of class A β-lactamases, evidence of additional slow motions on the µs-ms timescale was extracted from model-free calculations. This is consistent with the greater number of resonances that could not be assigned in this chimera relative to the parental β-lactamases, and is consistent with this well-folded and functional chimeric β-lactamase displaying increased slow time-scale motions.
- Published
- 2012
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.