BIOL368/S20:Sahil Patel Week 11

Purpose

The purpose of this assignment is to critically analyze a primary research article in order to help facilitate the formation of our final research question as well as to share with the class in a journal club.

Key Terms/Definitions

Glycoprotein: Any of the conjugated proteins, characterized by having one or more covalently-linked carbohydrate residues. (https://www.biologyonline.com/dictionary/glycoprotein)
cryo–electron microscopy: An electron microscopic technique that involves freezing the biological sample in order to view the sample with the least possible distortion and the fewest possible artifacts. (Shiel, 2018)
Antibody: An immunoglobulin, a specialized immune protein, produced because of the introduction of an antigen into the body, and which possesses the remarkable ability to combine with the very antigen that triggered its production. (Shiel, 2018)
Pathogen: An agent causing disease or illness to its host, such as an organism or infectious particle capable of producing a disease in another organism. (https://www.biologyonline.com/dictionary/pathogen)
Metastable: a state in which matter experiences a local equilibrium, but will be disturbed by a small perturbation resulting in a move to a lower energy state. (http://soft-matter.seas.harvard.edu/index.php/Metastable)
Recombinant: A cell or an individual with a new combination of genes not found together in either parent, usually applied to linked genes. (https://www.biologyonline.com/dictionary/recombinant)
Protomer: Any of the subunits constituting a larger structure, such as any of the polypeptide chains in an oligomeric protein. (https://www.biologyonline.com/dictionary/protomer)
Furin: subtilisin like eukaryotic endopeptidase with substrate specificity for consensus sequence arg X Lys/Arg arg at the cleavage site. (https://www.biologyonline.com/dictionary/furin)
Cross-reactive antibody: antibodies which don’t respond to any one specific antigen, but will respond to a number of them. These antibodies can be responsible for false positive results in antigen–antibody tests. (https://www.biologyonline.com/dictionary/cross-reactive-antibody)
Epitope: That part of an antigenic molecule to which the t-cell receptor responds, a site on a large molecule against which an antibody will be produced and to which it will bind. (https://www.biologyonline.com/dictionary/epitope)

Article Outline

What is the importance or significance of this work?
- In response to the global outbreak of the coronavirus and the panic caused by the pandemic, this work provides valuable information, including insight on the structure of the spike in the prefusion conformation of the 2019-nCoV, to help guide the development of vaccines/drugs for the novel coronavirus.

What were the limitations in previous studies that led them to perform this work?
- No previous studies had been performed on the 2019-nCoV.

How did they overcome these limitations?
- They overcame these limitations by first examining the structure of the virus, which was possible due to the available sequence data. By comparing this to the SARS-CoV that caused the outbreak in 2004, they were able to make key observations in relating structure to function.

What is the main result presented in this paper?
- The main result of the paper was discovering the structural data of the 2019-nCoV. The atomic-level structure of the spike will guide protein-engineering efforts which will improve antigenicity and protein expression, thereby facilitating vaccine development.

What were the methods used in the study?
- Protein expression and purification to express the prefusion S ectodomain
- Cryo-Electron Microscopy sample preparation, data collection, and data processing
- Sequence alignment of 2019-nCoV S, SARS-CoV S and RaTG13 S.

Briefly state the result shown in each of the figures and tables.
- Figure 1A: Structure of 2019-nCoV S in the prefusion conformation colored based on domain.
- Figure 1B: The prefusion structure of the 2019-nCoV S protein with a single RBD in the up conformation. The two RBD down protomers are shown using cryo-EM density in either white or gray and the RBD up protomer is shown in ribbons colored corresponding to the schematic in Figure 1A.
- Figure 2A: Structures of 2019 nCoV S RBD down protomer (left), 2019 nCoV S RBD up protomer (center), SARS-CoV S RBD up protomer (right).
- Figure 2B: The receptor-binding domains of the 2019-nCoV and SARS-CoV were aligned depending on locality of adjacent N Terminal Domain from neighboring protomer, displaying the 2019-nCoV RBD (green), SARS-CoV RBD (white) and the 2019-nCoV NTD (blue).
- Figure 2C: NTD (top left), RBD (top right), SD1&SD2 (bottom left), and S2 (bottom right). Structural comparison shows structural homology between the proteins.
- Figure 3A: Shows that ACE2 binds to 2019-nCoV S ectodomain with an affinity 10 to 20 times higher than when it binds to SARS-CoV S.
- Figure 3B: Diagram depicts 2019-nCoV S protein bound with ACE2 (blue) and S protein protomers colored tan, pink, and green.
- Figure 4A: Shows shared receptor usage between SARS-CoV S and 2019-nCoV S.
- Figure 4B: Biolayer interferometry sensorgram to show how ACE2 binds to 2019-nCoV RBD-SD1.
- Figure 4C: Biolayer interferometry used to make graphs showing cross-reactivity of the SARS-CoV RBD-directed antibodies.

How do the results of this study compare to the results of previous studies?
- The previous studies done have not included the novel coronavirus, thus it is a novel study which will play an important role as an initial step in the right direction.

What are the important implications of this work?
- This work allows for a better understanding of the binding affinity of the RBD and ACE2 which will help guide further research.

What future directions should the authors take?
- Future directions should focus on applying these structural findings in clinical research in order to develop medical countermeasures to the virus.

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 think that this paper was a critical first step. One of the most common themes in biological sciences is the relationship between structure and function. So, now that we have a better understanding of the structure, we will be able to make greater strides in uncovering more information about the 2019 nCoV.

Conclusion

By presenting and outlining this primary research article, we were able to effectively shift our focus of the class from HIV-1 gp120 structure and function to the more currently demanding field of research that is, the novel coronavirus. The article that our group read and presented during our journal club mainly discussed the structural comparison of the SARS CoV of 2004 and the 2019 nCoV, as well as uncovering their difference in binding affinity to ACE2. Overall, this article is one of many to come as this is a topic of utmost importance.

Acknowledgments

I contacted my homework partners Annika and Jenny during the week via text as well as over zoom to discuss the assignment as well as our journal club presentation.
Outline questions were copied from the Week 11 assignment page.
Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

References

Glycoprotein Definition and Examples - Biology Online Dictionary. (2020, January 28). Retrieved April 15, 2020, from https://www.biologyonline.com/dictionary/glycoprotein
Shiel, W. C. (2018, December 21). Definition of Cryo-electron microscopy. Retrieved April 15, 2020, from https://www.medicinenet.com/script/main/art.asp?articlekey=24623
Shiel, W. C. (2018, December 11). Definition of Antibody. Retrieved from https://www.medicinenet.com/script/main/art.asp?articlekey=19101
Pathogen Definition and Examples - Biology Online Dictionary. (2020, January 29). Retrieved April 15, 2020, from https://www.biologyonline.com/dictionary/pathogen
Metastable. (2009, December 3). Retrieved April 15, 2020, from http://soft-matter.seas.harvard.edu/index.php/Metastable
Recombinant Definition and Examples - Biology Online Dictionary. (2019, October 7). Retrieved April 15, 2020, from https://www.biologyonline.com/dictionary/recombinant
Protomer Definition and Examples - Biology Online Dictionary. (2019, October 7). Retrieved April 15, 2020, from https://www.biologyonline.com/dictionary/protomer
Furin Definition and Examples - Biology Online Dictionary. (2019, October 7). Retrieved April 15, 2020, from https://www.biologyonline.com/dictionary/furin
Cross-reactive antibody Definition and Examples - Biology Online Dictionary. (2019, October 7). Retrieved April 15, 2020, from https://www.biologyonline.com/dictionary/cross-reactive-antibody
Epitope Definition and Examples - Biology Online Dictionary. (2019, October 7). Retrieved April 15, 2020, from https://www.biologyonline.com/dictionary/epitope
OpenWetWare. (2020). BIOL368/S20:Week 11. Retrieved April 15, 2020, from https://openwetware.org/wiki/BIOL368/S20:Week_11.
Wrapp, D., Wang, N., Corbett, K. S., Goldsmith, J. A., Hsieh, C. L., Abiona, O., ... & McLellan, J. S. (2020). Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science, 367(6483), 1260-1263. DOI: 10.1126/science.abb2507