Carolyne week 11

From OpenWetWare
Jump to navigationJump to search

Purpose

The purpose of this activity is to understand how the structure of receptor binding domain in SARS-CoV-2 allows ACE2 receptors to recognize the virus.

Biological Terms

  1. Residue: any of the incorporated amino‐acid moieties in a peptide or protein (Cammack et. al., 2008)
  2. Ferredoxin(s): small proteins that contains one or more iron-sulphur clusters and are generally involved in electron transfer (Lackie and Nation, 2019)
  3. Homodimer: a protein made up of paired identical polypeptides (King et. al., 2014)
  4. Ternary complex: any complex formed between three chemical entities (Cammack et. al., 2008)
  5. FLAG: a proprietary synthetic epitope tag consisting of eight amino acids and readily detected using anti‐FLAG antibodies (Cammack et. al., 2008)
  6. Heterodimer: any molecular structure in which two nonidentical subunits are associated (Cammack et. al., 2008)
  7. Peptidase: an enzyme catalyzing the hydrolytic cleavage of peptide bonds (King et. al., 2014)
  8. Antiparallel: describing of a pair of parallel linear structures having directional polarity or asymmetry in opposite directions (Cammack et. al., 2008)
  9. Protomers: single polypeptide chains (either identical or nonidentical) of a multimeric protein (King et. al., 2014)
  10. Enteric: describing something within the intestine, or a substance given by this route (Lackie and Nation, 2019)

Article Outline

Introduction

  • What is the importance or significance of this work?
    • SARS-CoV-2 has caused an epidemic (now pandemic) of coronavirus disease 2019(COVID-19) around the world. By February 2020, more than 2000 people had died of COVID-19. The genome of SARS-CoV-2 is 80% identical to the genome of SARS-CoV, another virus that like SARS-COV-2 causes severe respiratory issues in humans.
    • SARS-CoV binds to the peptidase domain (PD) inACE2 using its spike glycoprotein (S protein). Specifically the S1 subunit since it contains the receptor binding domain (RBD) that is able to interact with ACE2. A previous study showed that the S protein of SARS-COV-2 was able to bind to ACE2 as well.
    • In order for SARS-CoV to be able to fuse to the host with the S2 subunit, the S1 subunit needs to successfully bind to ACE2.
  • What were the limitations in previous studies that led them to perform this work?
    • Previous studies that looked at the structure of ACE2 in complex with the S protein or the receptor binding domain (RBD) of SARS-CoV only had information about the structure of the peptidase domain (PD) of ACE2. This is because ACE2 has a transmembrane helix that made it difficult to figure out the full-structure.
  • How did they overcome these limitations?
    • They realized that ACE2 also chaperones another protein, BAT1. However, the interactions between the structures of ACE2 and BAT1 were not clear. The researchers previously were able to figure out the full structure of the LAT1-4F2hc complex. They figured that since ACE2 and BAT1 have a similar membrane tracking mechanism, they could resolve the full structure of ACE 2 and figure out how it might interact with SARS-CoV-2.

Methods

  • What were the methods used in the study?
    • The researchers used cryo-electron microscopy (cryo-EM) to determine the structures of ACE2 and BAT1. They tagged the proteins with FLAG and Strep and then express them in human embryonic kidney cells. They used size exclusion chromatography and affinity resin to purify the proteins. The samples were then prepared for cryo-EM and the cryo-EM data was used to create 3D reconstructions of the proteins.
    • The researchers also used cryo-EM to determine the structure of the SARS-CoV-2 RBD-ACE2-BAT1 complex. They mixed purified ACE2-BAT1 with purified RBD from SARS-COV-2. The mixture was prepared for cryo-EM and the images were reconstructed in 3D to determine the structure of the RBD-ACE2-BAT1 complex.
    • They compared ACE2 binding of SARS-CoV and SARS-CoV-2 by superimposing an image of the RBD of SARS-CoV and ACE2 PD complex onto the image they made of the SARS-CoV-2 RBD-ACE2-BAT1 complex.

Results

  • What is the main result presented in this paper?
    • The researchers resolved the full structure of the ACE2-BAT1 receptor complex and showed that two S proteins in SARS-CoV-2 can bind to one ACE2 homodimer.
  • Briefly state the result shown in each of the figures and tables, not just the ones you are presenting.
    • Figure 1: Figure 1 has four parts. In part A, they show a graph produced from the size exclusion chromatography. The graph shows the sample is pure because the sample produced only one, narrow peak at a UV absorbance of 300. Part B shows the 3D reconstruction of the ACE2-BAT1 complex at a 2.9 angstrom resolution. This shows the individual dimers in the heterodimer complex. In Part C, a cartoon model is used to represent the dimers, shows which residues in ACE2 are part of the domains in the protein, and show how ACE2 and BAT1 interact. It also shows the PD regions in ACE2 contribute to dimer formation. In part D, they show that the PDs in ACE2 are separated in this reconstruction.
      • Takeaway: Together, the researchers used this information to establish that the ACE2-BAT1 complex has two conformations, an open conformation and a closed conformation. It also shows that the rotation of the PD domains are responsible for changing the conformation.
    • Figure 2: Part A shows that the neck domain is mostly responsible for ACE2 dimer formation. Part B shows the residues in the neck domain, revealing that there is variety of polar interactions that make the dimer stable. Part C shows the residues in the PD interface, revealing that there are still polar interactions present but they are much weaker compared to the neck domain. Part D shows the distance between the PDs in the homodimer is about 25 angstroms in the open conformation.
      • Takeaway: Together, this information shows that ACE2 forms a homodimer. The stability of the homodimer comes from polar interactions in the neck domain, but the weak interactions in the PD allow the dimer to switch between open and closed conformations.
    • Figure 3: Part A of this image shows the cyro-EM map of the SARS-COV-2 RBD complexed with the ACE2-BAT1. They only gained the structure of the closed ACE2 conformation when it is complexed with RBD and BAT1. In part B, they show a structure model of the RBD-ACE2-BAT1 complex. This model shows that each PD can bind to 1 RBD.
      • Takeaway: The RBD in SARS-CoV-2 is able to bind to the PD in ACE2. One ACE2 homodimer is able to bind to two RBDs.
    • Figure 4: In part A of this figure, they show the binding interaction between the interface of the RBD in SARS-CoV-2 and the PD in ACE2. They find that the important structures in SARS-CoV-2 are the al helix, a2 helix, and a loop connecting B3 and B4 antiparallel strands. Parts B, C, and D focus on various regions in the binding interaction so they can study the residues in ACE2 that interact with the residues in SARS-CoV-2. Part B focuses on the N terminus, part C focuses on the bridge segment, and part D focuses on the C terminus of the RBD.
      • Takeaway: The RBD interacts with ACE2 through the al helix, a2 helix, and a loop connecting B3 and B4 antiparallel strands. This interaction features many polar residues and is stabilized by hydrogen bonds and van der Waals forces.
    • Figure 5: Part A shows the a comparison of the binding interface between SARS-CoV and ACE2 and SARS-CoV-2 and ACE2. The images are superimposed on each other so it is easy to see structural variation. Parts B, C, and D look at the N terminus, bridge, and C terminus of the RBD to observe differences in residue identity and residue interaction between the two viruses.
      • Takeaway: Through the viruses show a high degree of similarity in their interactions with ACE2, there are sequence variations between the viruses that may affect how they interact with ACE2.

Discussion and Implications

  • How do the results of this study compare to the results of previous studies.
    • A previous study showed the SARS-CoV-2 S protein is trimeric and has two RBDs in a down conformation and one in an up conformation. The researchers were able to use their results to confirm that binding is only possible with the RBD in the up conformation.
    • Another study showed that BAT1 may interact with a coronavirus receptor. The structure in this study suggests that BAT1 may be able to block the S protein from cleaving ACE2 effectively, which might help to inhibit viral entry into a cell.
  • What are the important implications of this work?
    • Having a full-structure of the ACE2 receptor is important because now people can use the structure to design antibodies or other drugs that block SARS-COV-2. It also will help researchers to have a better understanding of the actual steps that allow the ACE2 to recognize SARS-CoV-2 and better compare SARS-CoV-2 to SARS-CoV.
  • What future directions should the authors take?
    • Based on the results they showed, it would be interesting to see if the authors or another research group can use the structural information to design a drug or antibody that can block the RBD-ACE2-BAT1 interaction.
  • Give a critical evaluation of how well you think the authors supported their conclusions with the data they showed. Are there any limitations or major flaws to the paper?
    • Overall, I think the authors provided strong evidence to support their findings. The images clearly show the structure of ACE2 and BAT1, and how this complex interacts with the RBD in SARS-CoV-2. They also have strong evidence to show how differences in the binding between SARS-CoV and ACE2 versus SARS-CoV-2 and ACE2 can explain why the viruses may have different affinities for ACE2. As the authors state, however, this study doesn't account for any cofactors or other interactions that may be important for SARS-CoV-2 to bind to ACE2. So these results are a starting point, but not the complete picture.

Scientific Conclusion

ACE2 forms a homodimer through two PD domain interface and the neck domain interface. The PD domain of ACE2 is a dimeric protein interacts with the RBD of SARS-CoV-2 that is in the up conformation. The primary part of the RBD that help SARS-COV-2 to bind is the a1 helix, while other parts (a2 helix, linker) play smaller roles. Overall, SARS-CoV and SARS-CoV-2 have similar interactions with ACE2. But possible differences in their affinity for ACE2 can be explained because they have different amino acids that can enable the RBD to bind more or less tightly. This structure information will allow researchers to understand how SARS-COV-2 binds to ACE2 and potentially design inhibitors to block this interaction.

Presentation Link

Acknowledgements

I copied the questions from the Week 11 assignment page and used them to determine what content should be in the outline. I copied the reference for the Yan et. al. (2020) article onto this page from the Week 11 assignment page. I worked with Jack and Karina to create the journal club presentation over Google Slides. Except for what is noted above, this individual journal entry was completed by me and not copied from another source. Carolyne (talk) 22:42, 15 April 2020 (PDT)

References

  1. Yan, R., Zhang, Y., Li, Y., Xia, L., Guo, Y., & Zhou, Q. (2020). Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science, 367(6485), 1444-1448. doi: 10.1126/science.abb2762
  2. OpenWetWare. (2020). BIOL368/S20:Week 11. Retrieved April 2, 2020, from https://openwetware.org/wiki/BIOL368/S20:Week_11.
  3. King, R., Mulligan, P., & Stansfield, W. (2014). homodimer. In A Dictionary of Genetics. : Oxford University Press. Retrieved 15 Apr. 2020, from https://electra.lmu.edu:5305/view/10.1093/acref/9780199766444.001.0001/acref-9780199766444-e-3102?rskey=acW2Fy&result=1.
  4. King, R., Mulligan, P., & Stansfield, W. (2014). protomer. In A Dictionary of Genetics. : Oxford University Press. Retrieved 15 Apr. 2020, from https://electra.lmu.edu:5305/view/10.1093/acref/9780199766444.001.0001/acref-9780199766444-e-5516?rskey=QiCwcK&result=1.
  5. King, R., Mulligan, P., & Stansfield, W. (2014). peptidase. In A Dictionary of Genetics. : Oxford University Press. Retrieved 15 Apr. 2020, from https://electra.lmu.edu:5305/view/10.1093/acref/9780199766444.001.0001/acref-9780199766444-e-4973?rskey=0HtseN&result=1.
  6. Cammack, R., Atwood, T., Campbell, P, Parish, H., Smith, A., Vella, F., & Stirling, J. (2008). residue. In Oxford Dictionary of Biochemistry and Molecular Biology (2 ed.) : Oxford University Press. Retrieved 15 Apr. 2020, from https://electra.lmu.edu:5305/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-17205?rskey=09WZtc&result=2.
  7. Cammack, R., Atwood, T., Campbell, P, Parish, H., Smith, A., Vella, F., & Stirling, J. (2008). FLAG. In Oxford Dictionary of Biochemistry and Molecular Biology (2 ed.) : Oxford University Press. Retrieved 15 Apr. 2020, from https://electra.lmu.edu:5305/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-7087?rskey=98xsQG&result=5.
  8. Cammack, R., Atwood, T., Campbell, P, Parish, H., Smith, A., Vella, F., & Stirling, J. (2008). ternary complex. In Oxford Dictionary of Biochemistry and Molecular Biology (2 ed.) : Oxford University Press. Retrieved 15 Apr. 2020, from https://electra.lmu.edu:5305/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-19337?rskey=57dIda&result=1.
  9. Cammack, R., Atwood, T., Campbell, P, Parish, H., Smith, A., Vella, F., & Stirling, J. (2008). heterodimer. In Oxford Dictionary of Biochemistry and Molecular Biology (2 ed.) : Oxford University Press. Retrieved 15 Apr. 2020, from https://electra.lmu.edu:5305/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-8897?rskey=QFqA3l&result=1.
  10. Cammack, R., Atwood, T., Campbell, P, Parish, H., Smith, A., Vella, F., & Stirling, J. (2008). antiparallel. In Oxford Dictionary of Biochemistry and Molecular Biology (2 ed.) : Oxford University Press. Retrieved 15 Apr. 2020, from https://electra.lmu.edu:5305/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-1326?rskey=9ApNbQ&result=3.
  11. Lackie, J. and Nation, B. (2019). ferredoxins. In A Dictionary of Biomedicine (2 ed.) : Oxford University Press. Retrieved 15 Apr. 2020, from https://electra.lmu.edu:5305/view/10.1093/acref/9780191829116.001.0001/acref-9780191829116-e-3479?rskey=ImL0OJ&result=1
  12. Lackie, J. and Nation, B. (2019). enteric. In A Dictionary of Biomedicine (2 ed.) : Oxford University Press. Retrieved 15 Apr. 2020, from https://electra.lmu.edu:5305/view/10.1093/acref/9780191829116.001.0001/acref-9780191829116-e-3157?rskey=K6WHUs&result=3

User Page and Template Links

Individual Journal Pages

  1. Carolyne week 2
  2. Carolyne week 3
  3. Carolyne week 4
  4. Carolyne week 5
  5. Carolyne week 6
  6. Carolyne week 8
  7. Carolyne week 9
  8. Carolyne week 10
  9. Carolyne week 11
  10. Carolyne week 13
  11. Carolyne week 14

Weekly Assignments

  1. BIOL368/S20:Week 1
  2. BIOL368/S20:Week 2
  3. BIOL368/S20:Week 3
  4. BIOL368/S20:Week 4
  5. BIOL368/S20:Week 5
  6. BIOL368/S20:Week 6
  7. BIOL368/S20:Week 8
  8. BIOL368/S20:Week 9
  9. BIOL368/S20:Week 10
  10. BIOL368/S20:Week 11
  11. BIOL368/S20:Week 13
  12. BIOL368/S20:Week 14

Class Journal Pages

  1. BIOL368/S20:Class Journal Week 1
  2. BIOL368/S20:Class Journal Week 2
  3. BIOL368/S20:Class Journal Week 3
  4. BIOL368/S20:Class Journal Week 4
  5. BIOL368/S20:Class Journal Week 5
  6. BIOL368/S20:Class Journal Week 6
  7. BIOL368/S20:Class Journal Week 8
  8. BIOL368/S20:Class Journal Week 9
  9. BIOL368/S20:Class Journal Week 10
  10. BIOL368/S20:Class Journal Week 11
  11. BIOL368/S20:Class Journal Week 13
  12. BIOL368/S20:Class Journal Week 14
Retrieved from "https://openwetware.org/mediawiki/index.php?title=Carolyne_week_11&oldid=1081285"