Using single-molecule FRET analysis, a team of researchers has revealed how the different conformations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein are interconnected. Figuring out the different conformations in real-time and which conformations antibodies prefer may help design effective vaccines and drugs. The research is published on the preprint server bioRxiv*.
Illustrations 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.
SARS-CoV-2 illustration. Image Credit: Orpheus FX / Shutterstock
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.
This transmission electron microscope image shows SARS-CoV-2—also known as 2019-nCoV, the virus that causes COVID-19—isolated from a patient in the U.S. Virus particles are shown emerging from the surface of cells cultured in the lab. The spikes on the outer edge of the virus particles give coronaviruses their name, crown-like. Image captured and colorized at NIAID's Rocky Mountain Laboratories (RML) in Hamilton, Montana. Credit: NIAID
Four different conformations of spike protein
To understand the different conformations in real-time, a team of researchers used single-molecule Förster resonance energy transfer (FRET). 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.
Using available structures of the spike protein, the researchers labeled sites with fluorophores that can be used to detect the distance changes between the fluorophores with changes in the conformations. The team excited the fluorescent-labeled proteins using green laser and recorded the emission.
The researchers found low (~0.1), intermediate (~0.3 and ~0.5), and high (~0.8) FRET efficiencies. They say 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 authors say the intermediate conformation corresponds to all the RBDs pointing downward, toward the virus surface. They found that a disulfide bridge between two amino acids stabilizes the spike protein, which, based on previous studies, corresponds to the downward orientation.
When they 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.
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.
Thus, 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.
Antibodies have at least two neutralization mechanisms
Next, the team analyzed 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.
The antibodies from the other patient stabilized the RBD-down conformation. Although the antibodies thwarted binding of the hACE2 receptor, “but RBD competition did not affect its recognition of S, suggesting that its neutralization activity does not solely rely on blocking the receptor interface,” write the authors.
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.
Hence, strategies for designing effective vaccines or drugs can use different approaches for binding to the SARS-CoV-2 spike protein.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.