The force of sugar in the SARS-CoV-2 spike/ACE-2 interaction

Virusreceptor interactions are pivotal in establishing an infection. Viruses, particularly RNA viruses, such as SARS-CoV-2, constantly evolve and any mutations in the viral spike surface glycoprotein, particularly in the receptor-binding domain, need to be thoroughly monitored. Also, the port of entry for SARS-CoV-2, the receptor ACE-2 displays heterogeneity among humans. The viral spike protein and the human receptor are heavily glycosylated and glycans indirectly support or are directly involved in the interaction of the two proteins. Any mutations in the viral spike protein or ACE-2 single nucleotide polymorphisms that result in the loss of a strategically-positioned glycan in or close to the binding interface therefore require our attention, as these may in the worst case increase susceptibility to viral infection and transmissibility. A profound understanding of viralreceptor interactions is of significant importance, allowing us to expand our knowledge of tissue and species tropism, pathogenesis in certain human populations and early preparedness for emerging variants of concerns. The overall aim of this project is to produce and analyze different spike and ACE-2 variants with site- specific ablations of glycosylation sites within the binding interface. In contrast to previous studies using already emerged spike variants and the wildtype receptor ACE-2, we put a special focus on specific glycosylation sites of the interaction partners, that, owing to their proximal/strategic position, are suggested to affect viral-host interaction and naturally occurring ACE-2 glycovariant polymorphisms. We will evaluate the capability of the panel of interaction partners to bind under dynamic conditions and quantify binding strengths and kinetics, as well as map their interaction energy landscape. In addition, we will monitor the structural role of the spike glycomutations and their involvement in modulating its conformational dynamics and the inhibitory effect of soluble ACE-2 glycovariants on the binding of spike glycomutants to cellular ACE-2 receptors. Soluble SARS-CoV-2 spike and ACE-2 glycomutants will be recombinantly expressed in human cell lines and extensively characterized by quantitative glycopeptide analysis. The interactions between them will be analysed in single molecule and cell force spectroscopy experiments that, unlike ensemble methods, can capture every individual binding-unbinding events. High speed AFM movies will directly film dynamic conformational changes of isolated SARS-CoV-2 spike protein glycomutants. Our comprehensive investigations will not only result in a valuable collection of data for deciphering the mechanisms of spike-ACE-2 variant interaction, but also provide an experimental basis for the design of novel therapeutics for effective blocking of viral variant entry.