Coronavirus Research: Study Led By Yale Reveals That Shape-Shifting SARS-CoV-2 Coronavirus Has Four Different Conformations Of Spike Protein

Coronavirus Research: Scientist from Yale University School Of Medicine, St. Jude Children's Research Hospital-Memphis, National Institutes of Health-Bethesda, Columbia University and  Montreal University-Canada using an imaging platform that utilizes single-molecule Förster resonance energy transfer (FRET) have discovered that the spike proteins of the SARS-CoV-2 coronavirus has 4 different conformations. This new discovery has implications for drug and vaccine developments.

The study results are published on a preprint server and is pending peer-review. https://www.biorxiv.org/content/10.1101/2020.09.10.286948v2
or https://www.biorxiv.org/content/10.1101/2020.09.10.286948v2.full.pdf
 
Previous studies had already shown that the SARS-CoV-2 coronavirus has shape-shifting capabilities to possible evade immune response. https://www.thailandmedical.news/news/breaking-covid-19-latest-study-reveals-that-sars-cov-2-spike-protein-has-shape-shifting-ability-to-evade-immune-attacks-and-to-ensure-its-survival
 
Typical depictions of the SARS-CoV-2 virus generally look like a spiky ball. The spikes poking out from the virus surface are called spike proteins (S). These spike proteins are what allows the virus to enter a host cell, causing infection.
 
The spike protein has two parts, a head (S1), sitting on a stick-like body (S2). The head binds to suitable receptors on the host cell using certain parts on it called the receptor-binding domains (RBDs). The stick-like body then causes the virus envelope to merge with the host cell membrane. The fusion of the virus to the human cell happens when the human angiotensin-converting enzyme 2 (hACE2) binds to the RBDs.
 
Studies have revealed that the spike protein can have different conformations. In one, in which all the RBDs are oriented downward so that these receptors are inaccessible for binding. A second, where one or two RBDs are oriented up, and a third orientation with all the RBDs are oriented up, where all the receptors are accessible. However, how these different conformations are interconnected and how they behave is yet unknown.
 
However this is the first detailed study to show that there are 4 conformations.
 
In order to study the different conformations in real-time, the study team used single-molecule Förster resonance energy transfer (FRET).
 
Typically in FRET, energy is transferred between two light-sensitive molecules. Since the efficiency of energy transfer is related to the distance between the two molecules, FRET is used to understand the distance between two parts of a molecule to which the light-sensitive molecules are attached.
 
Utilizing available structures of the spike protein, the study team labeled sites with fluorophores that can be used to detect the distance changes between the fluorophores with changes in the conformations. The study team then excited the fluorescent-labeled proteins using gre en laser and recorded the emission.
 
Significantly the team found low (~0.1), intermediate (~0.3 and ~0.5), and high (~0.8) FRET efficiencies. The team said this corresponds to at least four different conformations of the spike protein. The conformation with the intermediate FRET efficiency was the most abundant, based on counting several hundred FRET traces.
 
The study team said the intermediate conformation corresponds to all the RBDs pointing downward, toward the virus surface. The team found that a disulfide bridge between two amino acids stabilizes the spike protein, which, based on previous studies, corresponds to the downward orientation.
 
However when the team used the receptor hACE2 that binds to the RBDs, they found the low FRET state was more abundant, suggesting all the RBDs oriented in the upward direction, or away from the virus surface, when bound to the receptors.
 
Interestingly the spike protein was in equilibrium between the different conformations at physiological pH and room temperature. When the conformations changed, there was a specific order in which they transitioned; first from the low to the intermediate efficiency state, and then from the intermediate to the high-efficiency state.
 
Hence there is a specific sequence of activating structural transitions in the SARS-CoV-2 spike protein. The RBD-down conformation changes to the RBD-up conformation, which is activated by the receptor, via at least one intermediate conformation.
 
The study team next studied what happens to the conformations when antibodies bind. Using plasma from two convalescent patients, where the antibodies in the plasma bind to the spike protein, they found the spike protein to be in the RBD-up conformation for antibodies from one patient. This was similar to the orientation using the hACE2 receptor.
 
However the antibodies from the other patient stabilized the RBD-down conformation.
 
Despite the antibodies thwarting binding of the hACE2 receptor, “the RBD competition did not affect its recognition of S, suggesting that its neutralization activity does not solely rely on blocking the receptor interface,” commented the study team.
 
The team summarized, “Virus-associated S dynamically samples at least four distinct conformational states. In response to hACE2, S opens sequentially into the hACE2- bound S conformation through at least one on-path intermediate. Conformational preferences of convalescent plasma and antibodies suggest mechanisms of neutralization involving either competition with hACE2 for binding to RBD or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and conformations for immunogen design.”
 
Significantly the results suggest that the SARS-CoV-2 virus may be neutralized in different ways. One, by antibodies mimicking the hACE2 receptor and competing with it to bind with the spike protein; and two, by stabilizing the protein in the RBD-down conformation, preventing binding to the host cell.
 
The strategies could be utilized for designing effective vaccines or drugs that can use different approaches for binding to the SARS-CoV-2 spike protein.
 
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