Kvescio Week 11 Assignment

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Purpose

The purpose of this assignment was to evaluate an article about the ACE2 receptor binding in the SARS-CoV-2 virus, and to be able to discuss it with fellow students.

Definitions of Terms

Virion

  • a completed virus particle consisting of a nucleic acid core and a protein coat (King, Mulligan, Stansfield, 2014).

Angiotensin

  • A protein that has two forms: angiotensin I and angiotensin II. Angiotensin I is derived from a plasma protein and released by the action of renin from the kidneys (Kent, 2007).

Vasoconstriction

  • a decrease in the diameter of blood vessels, especially arteries (Martin and McFerran, 2017).

Transmembrane Protein

  • an integral membrane protein with the polypeptide chain exposed on both sides of the membrane (Lackie and Nation, 2019).

Homogeneity

  • The degree to which items are similar. This arises in describing the properties of a dataset: it measures the differences or similarities between the several studies (Ireland, 2010).

Glycosylation

  • The process in which a carbohydrate is joined to another molecule, such as a protein to form a glycoprotein (Hine, 2019).

Moiety

  • A characteristic part of a molecule. For example, in the ester C2H5COOCH3, the OCH3 can be regarded as the alcohol (methanol) moiety (King, Mulligan and Stansfield, 2014).

Protomer

  • A general term for the subunits that are assembled to form a larger structure. Cell biological examples are G-actin, which assembles to filamentous F-actin, and the tubulin heterodimer which is the subunit for microtubules. The latter example illustrates the utility of a term that does not assume protomers to be single molecules (Lackie, 2010).

Ferredoxin

  • A protein containing iron associated with sulphur that is a carrier molecule in the electron transport chain in photosynthesis . It accepts electrons from photosystem I and passes them on to NADP reductase, which donates them for the reduction of NADP + (Hine, 2019).

Enteric

  • of or pertaining to the intestine or gut (Cammack, Atwood, Campbell, Parish, Smith, Vella, Stirling, 2008).

Outline of Article

Introduction

  • What is the importance or significance of this work?
    • The importance of this work is to learn the secrets of SARS-CoV-02 that leads to the disease COVID-19, and is currently causing havoc across the world. The article evaluates the way the virus binds to the human receptor, providing insight into the molecular basis for SARS-CoV-02. Scientists can use this information to figure out how the virus works, and ultimately how to stop it.
  • What were the limitations in previous studies that led them to perform this work?
    • Previous studies reported the structure of the S protein of the virus and showed that the SARS-CoV-2 S protein bonds to the PD of the human receptor.
    • Dissociation constant (Kd) of binding was 15 nM.
    • Limitations of the previous studies involved lack of understanding of the full structure of the SARS-CoV-2 S protein.
    • Information about the structure of ACE2 (the human receptor) was limited to the PD domain.
    • The structure of the entire protein is difficult to confirm because of the single transmembrane (TM) helix of the human receptor (ACE2).
  • How did they overcome these limitations?
    • They discovered that the ACE2 has a function in which it is involved in membrane trafficking for B0AT1, or (SLC6A19).
    • B0AT1 is an amino acid transporter that mediates how amino acids go into the intestinal cells.
    • The trafficking mechanism for ACE2 and the amino acid transporter, B0AT1, is very similar to that of the LAT1-4F2hc complex.
    • The scientists were confident that the full length structure of the ACE2 will be revealed when it is working with B0AT1.

Methods

  • What were the methods used in the study?
      1. Cryo-electron microscopy (cryo-EM) was used to get the full lento structure of the ACE2-B0AT1 complex.
      2. The full length cells were tagged with Strep and FLAG tags on their N termini.
      3. After being tagged, the cells were co-expressed in a human kidney, and purified through resin and chromatography.
      4. The scientists then created a three-dimensional version of the selected particles.
      5. They then applied focused refinement and C2 symmetry expansion to get better resolution.
      6. The team continued to classify more particles and process them with resolution.
      7. Extensive interactions were mapped in order to confirm that there is stable dimer formation.
      8. 0.2 mg of purified RBD-mFc of SARS-CoV-2 was mixed with purified ACE2-B0AT1 complex.
      9. Cryo-grid preparation nd imaging was used to get a 3D image of the obtained complex.
      10. Focused refinement was applied to improve the resolution.

Results

  • Briefly state the result shown in each of the figures and tables, not just the ones you are presenting.
    • Figure 1
      • 1A: Sample has one narrow peak with an absorbance of 300. This indicates that the sample is pure.
      • 1B: Complex has a 2.9 resolution, shown in a three-dimensional figure.
      • 1C: Illustrates how ACE2 and B0AT1 interact with each other.
      • 1D: The PDs and ACE2 separate during the reconstruction.
    • Figure 2
      • 2A: Indicates the ACE2 dimer formation and that is it controlled by the neck domain.
      • 2B: there are residues in the neck domain. Dimer is stable.
      • 2C: There are residues in the PD. Interactions are quite weak.
      • 2D: There is a 25 angstrom distance between the PDs in the homodimer.
    • Figure 3
      • 3A: Researchers created a cyro-EM map of the virus when it interacts with the ACE2-BAT1 complex.
      • 3B: Model of RBD-ACE-B0AT1 complex.
    • Figure 4
      • 4A: The interaction of the PD in ACE2 and RBD in the novel virus.The structures are in a helix and connect in antiparallel strands.
      • 4B: The N terminus region of ACE2 and how it interacts with SARS-CoV-02
      • 4C: The interaction within the bridge segment.
      • 4D: The interaction in the RBD, specifically in the C terminus.
    • Figure 5
      • 5A: Compares the way the novel virus binds to the ACE2, and the way SARS-CoV interacted with the ACE2.
      • 5B: Shows how SARS-CoV-01 and SARS-CoV-02 differ and are similar in the N terminus.
      • 5C: Indicates the differences and similarities of the viruses in the bridge segment.
      • 5D: Indicates the differences and similarities of the viruses in the RBD, specifically in the C terminus.
  • What is the main result presented in this paper? (Hint: look at the last sentence of the introduction and restate it in plain English.)
    • Through cryo-electron microscopy, the researchers were able to discover the full length structure of the ACE2 in the presence of the transporter B0AT1. Two S proteins bind to one ACE2 homodimer in SARS-CoV-02.

Discussion

  • How do the results of this study compare to the results of previous studies?
    • In a previous study, the trimeric structure of the S protein of the virus was reported. One RBD was in an up conformation and two were in a down conformation. The study suggested that the PD clashes with the rest of the S protein when it is lined up to the RBD with a down conformation.
    • This study confirmed that there is indeed no clash when the complex is superimposed on the RBD.
    • Study confirmed that an up conformation of RBD is required to bind to ACE2.
  • What are the important implications of this work?
    • The study is incredibly important because it helps other researchers to better understand the structure of the virus. This way scientists can get closer to understanding the full structure and binding methods of SARS-CoV-02. Such work is important for developing a treatment, or vaccine against the virus.
  • What future directions should the authors take?
    • Researchers still need to examine interactions between the receptor (ACE2) and the virus SARS-CoV-2
    • They need to discover whether there is clustering between the receptor and the S proteins.
    • I would suggest continuing the study and using similar methods in order to examine the interaction between the dimeric ACE2 and the trimeric S proteins.
  • 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?
    • I believe that the paper was well written and very concise. The authors supported their conclusion well with the figures that they provided in the study. The study was a good starting off point in investigating the ACE2 receptor and how it binds in the novel virus. However, there is much more research that needs to be done in order to fully understand the ACE2 binding in SARS-CoV-02. There were no major flaws in the paper, however, there are still limitations in their results as to the progress of understanding the novel coronavirus.

Journal Club Presentation

Presentation

Scientific Conclusion

  • In all, the assignment was to get a better understanding of the novel virus, SARS-CoV-02, and to be able to evaluate an article with new scientific information on the topic. The article I was assigned studied the structure of the ACE2 receptor in humans, in order to evaluate how it binds to and interacts with SARS-CoV-02. The team was able to discover the full length of a certain region of the ACE2 receptor, however, there is still unknown knowledge about how it fully binds and interacts with the virus. The research done was a very crucial step in determining how to stop the virus that is currently a serious issue in the world. Further research will benefit from these findings, and scientists will be closer to developing a vaccine.

Acknowledgements

  • I copied and pasted the wiki syntax from Week 11.
  • I worked with Jack and Carolyn in Google Slides for our presentation.
  • I emailed Jack and Carolyn and asked them questions about our assignment.
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

(Kvescio (talk) 23:55, 15 April 2020 (PDT))

References

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