Interaction between Spike Protein of SARS-CoV-2 and Human Virus Receptor ACE2 Using Two-Color Fluorescence Cross-Correlation Spectroscopy

Abstract

Infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), is initiated by the interaction between a receptor protein, angiotensin-converting enzyme type 2 (ACE2) on the cell surface, and the viral spike (S) protein. This interaction is similar to the mechanism in SARS-CoV, a close relative of SARS-CoV-2, which was identified in 2003. Drugs and antibodies that inhibit the interaction between ACE2 and S proteins could be key therapeutic methods for preventing viral infection and replication in COVID-19. Here, we demonstrate the interaction between human ACE2 and a fragment of the S protein (S1 subunit) derived from SARS-CoV-2 and SARS-CoV using two-color fluorescence cross-correlation spectroscopy (FCCS), which can detect the interaction of fluorescently labeled proteins. The S1 subunit of SARS-CoV-2 interacted in solution with soluble ACE2, which lacks a transmembrane region, more strongly than that of SARS-CoV. Furthermore, one-to-one stoichiometry of the two proteins during the interaction was indicated. Thus, we propose that this FCCS-based interaction detection system can be used to analyze the interaction strengths of various mutants of the S1 subunit that have evolved during the worldwide pandemic, and also offers the opportunity to screen and evaluate the performance of drugs and antibodies that inhibit the interaction.

Tracking Heme-Protein Interactions in Healthy and Pathological Human Serum in Native Conditions by Miniaturized FFF-Multidetection

“…The amount of sample required for the analysis was extremely low, due to the exploitation of the miniaturized F4 variant, and the analysis time being below 30 min for each analysis. In agreement with pre-existing "single component" studies [29][30][31][32], we observed that serum's heme-scavenging power is mainly represented by SA and Hx, and to a much lower degree by some immunoglobulins. IgG scavenging contributions in the presence of clinically relevant free heme levels (5-40 µM) can instead be considered negligible.…”

“…In the case of serum-heme interactions, it is to be noted that the experimental difficulties are numerous, and the matrix is very complex. Consequently, all the experiments conducted up to date aimed at understanding the involvement of serum protein for heme scavenging were carried out in an isolated and compartmentalized way, which on one hand facilitates recognition, but on the other hand represent only a partial result that does not necessary fully translate to the real matrix or the physiological state [29][30][31][32]. To examine heme-protein interactions in a more representative scenario, where proteins are simultaneously present in the matrix (in their biological concentrations) and can both concur and compete for binding, it is necessary to develop a detection system that simplifies the sample while preserving its native state.…”

Correlation between In Vitro Neutralization Assay and Serological Tests for Protective Antibodies Detection

“…In addition, molecular dynamics simulations proved helpful in the prediction of new spike variants’ behaviors and the effectiveness of human neutralizing antibodies [ 24 ]. Furthermore, to develop a high-throughput platform to screen neutralizing antibodies against SARS-CoV-2, Fujimoto et al carried out two-color fluorescence cross-correlation spectroscopy experiments (FCCS), investigating the quantitative interactions between the spike protein and human soluble ACE2 receptor [ 25 ]. Conversely, Rusanen and co-workers labeled the viral spike and nucleoprotein antigens and incubated them with serum samples, developing a “mix and read” immunoassay based on Förster resonance energy transfer (FRET) [ 26 ].…”

Spike protein of the SARS-CoV-2 omicron variant interacts with actin

“…Recombinant proteins were expressed in murine neuroblastoma Neuro2a cells (CCL-131, ATCC, Manassas, VA, USA) and purified as previously reported [18].…”

“…FCCS measurements were performed using a ConfoCor 3 system combined with an LSM 510 META (Carl Zeiss, Jena, Germany) through a C-Apochromat 40×/1.2 NA Korr UV-VIS-IR water immersion objective (Carl Zeiss) as reported previously [18]. Briefly, the relative cross-correlation amplitude (RCA) was calculated as the following equation.…”