Jennymchua Week 11 Assignment

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Purpose

The purpose of this assignment is to learn the structure-function relationship of the 2019-nCoV spike in its prefusion conformation and to attempt to formulate a final research question using the guidance of the article.

Definitions

  • trimer
    • a molecular complex having three components or subunits (Cammack et al., 2008)
  • monoclonal antibody
    • an antibody produced artificially from a cell clone and therefore consisting of a single type of immunoglobulin (Martin and McFerren, 2017)
  • metastable
    • describing any body or system existing in a state of apparent equilibrium when undisturbed (Cammack et al., 2008)
  • stochasic
    • pertaining to an event or a process containing randomness or variability (King, Mulligan, and Stansfield, 2014)
  • ectodomain
    • the proteolytic release of extracellular domains of membrane-anchored proteins, provides a key regulatory mechanism of the signaling capacity of cell surface receptors (Arribas and Borroto, 2002)
  • protomer
    • any of the subunits of an oligomeric protein that are identical (Cammack et al., 2008)
  • hemagglutinin
    • any agglutinin of red blood cells (Cammack et al., 2008)
  • furin
    • a serine endopeptidase that processes various secretory proproteins (including some growth factors) by cleavage at paired basic amino acids (Lackie and Nation, 2019)
  • plasmon
    • a collective excitation for quantized oscillations of the electrons in a metal (King, Mulligan, and Stansfield, 2014)
  • ab initio
    • methods that make predictions about biological features using only a computational model without extrinsic comparison to existing data (Genscript, 2019)

Article outline

What is the importance or significance of this work?

Due to the pandemic state of the 2019 n-CoV outbreak, the understanding of the structure-function relationship of the spike in the prefusion conformation can improve antigenicity and protein expression for (crucial) vaccine development.

What were the limitations in previous studies that led them to perform this work?

While previous studies have tested three antibodies known to bind to the SARS-CoV spike protein, no other studies had tested the binding properties of the 2019-nCoV spike protein.

How did they overcome these limitations?

This study tested the binding properties of the current 2019-nCOV spike protein associated with the novel coronavirus causing the 2019-2020 global pandemic. They did this by using Ångstrom-resolution reconstruction and three-dimensional reconstruction in order to visualize how the spike glycoprotein binds to the human ACE2 enzyme and that it binds tighter than (SARS)-CoV.

What is the main result presented in this paper?

The 2019-nCoV spike protein binds at least ten times more tightly than the corresponding spike protein of SARS-CoV to the common host receptor, which can be helpful for further vaccine development.

What were the methods used in the study?

Essentially, two proline amino acid mutations were added to the glycoprotein spike of the 2019-nCoV virus which helps to stabilize binding. From there, the ectodomain (which includes the part of the protein that initiate contact with something else, i.e. the virus) was then purified and analyzed using Cryo-electron microscopy and Ångstrom-resolution reconstruction in order to visualize the conformations of the subunits, as well as the phase at which binding occurs and the movement of the host receptor that allows the protein to bind.

Briefly state the result shown in each of the figures and tables, not just the ones you are presenting.

  • Figure 1A
    • Primary structure of 2019-nCoV with various highlighted domains, including the RBD (receptor-binding domain) and the NTD (N-terminal domain) which are key in determining its antigenicity.
  • Figure 1B
    • Biophysical representation of 2019-nCoV in the up conformation. The RBD is highlighted as the part of the membrane that attaches to the host receptor.
  • Figure 2A
    • Shows the structural units of 2019-nCoV comparing the down and up conformations.
  • Figure 2B.
    • Receptor binding sites of 2019-nCoV and SARS-COV. Also shows the N-terminal domain to where 2019-nCoV binds to the host receptor. The major difference between SARS-CoV and 2019-nCoV is the position of the RBD in the down conformation. The RBD in 2019-nCoV angles closer to the central cavity of the trimer, while the RBD in SARS-CoV packs against the NTD. It is unknown what impact this makes.
  • Figure 3A
    • The binding kinetics of ACE2 to 2019-nCoV. As shown in the graph, the response occurs very fast and tapers slowly down over a span of approximately 700 seconds. While there is some legend on the side that shows numbers in units of meters per seconds (probably representative of kinetics and orders of reactions), it is unclear what each line necessarily represents and how those three numbers corresponds to the five lines.
  • Figure 3B
    • Staining representations and recreations of ACE2 binding to 2019-nCoV on top of SARS-CoV to show hwo closely they resemble each other structurally.
  • Figure 4A
    • Shows SARS-CoV RBD in white with the residues carried over to 2019-nCoV in red. In particular, the ACE2 binding site is shown circled in particular, as this is where the virus interacts with its host.
  • Figure 4B
    • Shows the antigenicity of of the 2019-nCoV binding site to ACE2. This is important because it fits the best fit trend 1:1 that was predicted.
  • Figure 4C
    • Shows similar binding data across three antibodies of 2019-nCoV virus. This is important because of how strong and tightly the virus binds with ACE2; this ultimately leads to little to no response of usable antibodies.

How do the results of this study compare to the results of previous studies?

The results of this study present a biophysical visualization of the 2019-nCoV spike protein as it binds to the host receptor and how tightly it does this. Previous studies examined the SARS-CoV virus, but this study builds upon that previous information and tries to compare both viruses to see a) if the same treatments can be used and/or b) where changes can be made in therapies used previously.

What are the important implications of this work?

As the novel coronavirus continues to spread, it continues to kill as well. There are no treatments, remedies, or medicinal preventatives currently, so understanding the mechanism(s) of how the virus interacts with the human host could be a key in developing a solution. Just as we learned in the HIV-1 unit of this class, there are many drugs that target various stages in the HIV-1 life cycle, so the findings from this study could help to work as a preventative or an early-stage treatment.

What future directions should the authors take?

With the information we know now about the binding affinity of the 2019-nCoV virus to a human host receptor, known vaccinations or medications that work on the affinity of ACE2 could be used in small trials to further vaccine development.

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?

This paper was very hard to read and a bit unorganized in terms of flow. However, they did provide clear structural evidence in their conclusions and findings with the data presented. Not only did they prove the binding affinity of 2019-nCoV to ACE2, but they provided comparisons with SARS-CoV, which is important in attempting to find treatments or solutions that did/didn't work with the epidemic associated with it in 2002. I don't see any major flaws or limitations, as the research on this virus is very new and not much is understood at this point.

Conclusion

This exercise helped reinforce the ideas learned when we first attempted Journal Club a few weeks ago in class. We were able to read a paper relevant to the ongoing coronavirus pandemic and understand how the figures/tables told the story of what question the researcher was attempting to answer in their work. For this instance, the biophysical properties of the 2019-nCoV glycoprotein spike were crucial in possible vaccine development, as it was shown to bind with a higher affinity than that of SARS-CoV. By comparing and contrasting the two viruses, we can rule out or re-consider possible treatment plans to curb the spread and fatalities.

Acknowledgments

  • I worked with my homework partners Annika and Sahil throughout the week via Zoom and text to work on the project.
  • Questions for the outline were copied and modified from the syntax available here.
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Jennymchua (talk) 16:38, 15 April 2020 (PDT)

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