ABSTRACT Viruses mutate under a variety of selection pressures, allowing them to continuously adapt to their hosts. Mutations in SARS-CoV-2 have shown effective evasion of population immunity and increased affinity to host factors, in particular to the cellular receptor ACE2. However, in the dynamic environment of the respiratory tract forces act on the binding partners, which raises the question whether not only affinity, but also force-stability of the SARS-CoV-2:ACE2 bond, might be a selection factor for mutations. Here, we use magnetic tweezers (MT) to study the effect of amino acid substitutions in variants of concern (VOCs) on RBD:ACE2 bond kinetics with and without external load. We find higher affinity for all VOCs compared to wt, in good agreement with previous affinity measurements in bulk. In contrast, Alpha is the only VOC that shows significantly higher force stability compared to wt. Investigating the RBD:ACE2 interactions with molecular dynamics simulations, we are able to rationalize the mechanistic molecular origins of this increase in force-stability. Our study emphasizes the diversity of contributions to the assertiveness of variants and establishes force-stability as one of several factors for fitness. Understanding fitness-advantages opens the possibility for prediction of likely mutations allowing rapid adjustment of therapeutics, vaccination, and intervention measures.

Mutations in SARS-CoV-2 have shown effective evasion of population immunity and increased affinity to the cellular receptor angiotensin-converting enzyme 2 (ACE2). However, in the dynamic environment of the respiratory tract, forces act on the binding partners, which raises the question of whether not only affinity but also force stability of the SARS-CoV-2-ACE2 interaction might be a selection factor for mutations. Using magnetic tweezers, we investigate the impact of amino acid substitutions in variants of concern (Alpha, Beta, Gamma and Delta) and on force-stability and bond kinetic of the receptor-binding domain-ACE2 interface at a single-molecule resolution. We find a higher affinity for all of the variants of concern (>fivefold) compared with the wild type. In contrast, Alpha is the only variant of concern that shows higher force stability (by 17%) compared with the wild type. Using molecular dynamics simulations, we rationalize the mechanistic molecular origins of this increase in force stability. Our study emphasizes the diversity of contributions to the transmissibility of variants and establishes force stability as one of the several factors for fitness. Understanding fitness advantages opens the possibility for the prediction of probable mutations, allowing a rapid adjustment of therapeutics, vaccines and intervention measures.

Significance In the dynamic environment of the airways, where SARS-CoV-2 infections are initiated by binding to human host receptor ACE2, mechanical stability of the viral attachment is a crucial fitness advantage. Using single-molecule force spectroscopy techniques, we mimic the effect of coughing and sneezing, thereby testing the force stability of SARS-CoV-2 RBD:ACE2 interaction under physiological conditions. Our results reveal a higher force stability of SARS-CoV-2 binding to ACE2 compared to SARS-CoV-1, causing a possible fitness advantage. Our assay is sensitive to blocking agents preventing RBD:ACE2 bond formation. It will thus provide a powerful approach to investigate the modes of action of neutralizing antibodies and other agents designed to block RBD binding to ACE2 that are currently developed as potential COVID-19 therapeutics.

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the trans-Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the D ' D3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose 2 buried cysteines, Cys1099 and Cys1142, that are vital for multimerization. By characterizing the conformational change at varying levels of force, we can quantify the kinetics of the transition and stability of the interface. We find a pronounced destabilization of the interface on lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the D ' D3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII, providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the D ' D3 domain in VWF biosynthesis and function, and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond.

Stretching and heating are everyday experiences for skin and tissue cells. They are also standard procedures to reduce the risk for injuries in physical exercise and to relieve muscle spasms in physiotherapy. Here, we ask which immediate and long-term mechanical effects of such treatments are quantitatively detectable on the level of individual living cells. Combining versatile optical stretcher techniques with a well-tested mathematical model for viscoelastic polymer networks, we investigate the thermomechanical properties of suspended cells with a photothermal rheometric protocol that can disentangle fast transient and slow 'inelastic' components in the nonlinear mechanical response. We find that a certain minimum strength and duration of combined stretching and heating is required to induce long-lived alterations of the mechanical state of the cells, which then respond qualitatively differently to mechanical tests than after weaker/shorter treatments or merely mechanical preconditioning alone. Our results suggest a viable protocol to search for intracellular biomolecular signatures of the mathematically detected dissimilar mechanical response modes.