Impact of the temperature on the interactions between common variants of the SARS-CoV-2 receptor binding domain and the human ACE2

Abstract

Several key mutations in the Spike protein receptor binding domain (RBD) have been identified to influence its affinity for the human Angiotensin-Converting Enzyme 2 (ACE2). Here, we perform a comparative study of the ACE2 binding to the wild type (Wuhan) RBD and some of its variants: Alpha B.1.1.7, Beta B.1.351, Delta B.1.617.2, Kappa B.1.617.1, B.1.1.7 + L452R and Omicron B.1.1.529. Using a coiled-coil mediated tethering approach of ACE2 in a novel surface plasmon resonance (SPR)-based assay, we measured interactions at different temperatures. Binding experiments at 10 °C enhanced the kinetic dissimilarities between the RBD variants and allowed a proper fit to a Langmuir 1:1 model with high accuracy and reproducibility, thus unraveling subtle differences within RBD mutants and ACE2 glycovariants. Our study emphasizes the importance of SPR-based assay parameters in the acquisition of biologically relevant data and offers a powerful tool to deepen our understanding of the role of the various RBD mutations in ACE2 interaction binding parameters.

Figure 1

Representation of our three-step SPR-based assay with capture of E5 coil tagged ACE2 (ACE2-E5) by a K5 coil surface.

Figure 2

SPR sensorgrams recorded for the RBD variants that do not contain the L452R mutation interacting with tethered ACE2 at different temperatures. The sensorgrams corresponding to variant injections at 10, 25 and 37 °C were globally fitted with a 1:1 Langmuir binding model (solid black lines). Residual plots are included underneath each sensorgram series.

Figure 3

SPR sensorgrams recorded for the RBD variants with the L452R mutation interacting with tethered ACE2 at different temperatures. The sensorgrams corresponding to variant injections at 10, 25 and 37 °C were globally fitted with a 1:1 Langmuir binding model (black solid lines). Residual plots are included underneath each sensorgram series.

Figure 4

Comparison of the affinity of the RBD/ACE2 interaction at 10 °C (in blue), 25 °C (in orange) and 37 °C (in red) for all RBD variants considered in this study. The fold difference with respect to the affinity of the wild type RBD at a given temperature is reported.