A tethered ligand assay to probe SARS-CoV-2:ACE2 interactions.

SignificanceIn 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.

Streptavidin/biotin: Tethering geometry defines unbinding mechanics.

Macromolecules tend to respond to applied forces in many different ways. Chemistry at high shear forces can be intriguing, with relatively soft bonds becoming very stiff in specific force-loading geometries. Largely used in bionanotechnology, an important case is the streptavidin (SA)/biotin interaction. Although SA's four subunits have the same affinity, we find that the forces required to break the SA/biotin bond depend strongly on the attachment geometry. With AFM-based single-molecule force spectroscopy (SMFS), we measured unbinding forces of biotin from different SA subunits to range from 100 to more than 400 pN. Using a wide-sampling approach, we carried out hundreds of all-atom steered molecular dynamics (SMD) simulations for the entire system, including molecular linkers. Our strategy revealed the molecular mechanism that causes a fourfold difference in mechanical stability: Certain force-loading geometries induce conformational changes in SA's binding pocket lowering the energy barrier, which biotin has to overcome to escape the pocket.

Single-Molecule Manipulation in Zero-Mode Waveguides.

The mechanobiology of receptor-ligand interactions and force-induced enzymatic turnover can be revealed by simultaneous measurements of force response and fluorescence. Investigations at physiologically relevant high labeled substrate concentrations require total internal reflection fluorescence microscopy or zero mode waveguides (ZMWs), which are difficult to combine with atomic force microscopy (AFM). A fully automatized workflow is established to manipulate single molecules inside ZMWs autonomously with noninvasive cantilever tip localization. A protein model system comprising a receptor-ligand pair of streptavidin blocked with a biotin-tagged ligand is introduced. The ligand is pulled out of streptavidin by an AFM cantilever leaving the receptor vacant for reoccupation by freely diffusing fluorescently labeled biotin, which can be detected in single-molecule fluorescence concurrently to study rebinding rates. This work illustrates the potential of the seamless fusion of these two powerful single-molecule techniques.

Switchable reinforced streptavidin.

The complex of the small molecule biotin and the homotetrameric protein streptavidin is key to a broad range of biotechnological applications. Therefore, the behavior of this extraordinarily high-affinity interaction under mechanical force is intensively studied by single-molecule force spectroscopy. Recently, steered molecular dynamics simulations have identified a low force pathway for the dissociation of biotin from streptavidin, which involves partial unfolding of the N-terminal ß-sheet structure of monovalent streptavidin's functional subunit. Based on these results, we now introduced two mutations (T18C,A33C) in the functional subunit of monovalent streptavidin to establish a switchable connection (disulfide bridge) between the first two ß-strands to prevent this unfolding. In atomic force microscopy-based single-molecule force spectroscopy experiments, we observed unbinding forces of about 350 pN (at a force-loading rate of 10 nN s-1) for pulling a single biotin out of an N-terminally anchored monovalent streptavidin binding pocket - about 1.5-fold higher compared with what has been reported for N-terminal force loading of native monovalent streptavidin. Upon addition of a reducing agent, the unbinding forces dropped back to 200 pN, as the disulfide bridge was destroyed. Switching from reducing to oxidizing buffer conditions, the inverse effect was observed. Our work illustrates how the mechanics of a receptor-ligand system can be tuned by engineering the receptor protein far off the ligand-binding pocket.

Dronpa: A Light-Switchable Fluorescent Protein for Opto-Biomechanics.

Since the development of the green fluorescent protein, fluorescent proteins (FP) are indispensable tools in molecular biology. Some FPs change their structure under illumination, which affects their interaction with other biomolecules or proteins. In particular, FPs that are able to form switchable dimers became an important tool in the field of optogenetics. They are widely used for the investigation of signaling pathways, the control of surface recruitment, as well as enzyme and gene regulation. However, optogenetics did not yet develop tools for the investigation of biomechanical processes. This could be leveraged if one could find a light-switchable FP dimer that is able to withstand sufficiently high forces. In this work, we measure the rupture force of the switchable interface in pdDronpa1.2 dimers using atomic force microscopy-based single molecule force spectroscopy. The most probable dimer rupture force amounts to around 80 pN at a pulling speed of 1600 nm/s. After switching of the dimer using illumination at 488 nm, there are hardly any measurable interface interactions, which indicates the successful dissociation of the dimers. Hence this Dronpa dimer could expand the current toolbox in optogenetics with new opto-biomechanical applications like the control of tension in adhesion processes.

Direction Matters: Monovalent Streptavidin/Biotin Complex under Load.

Novel site-specific attachment strategies combined with improvements of computational resources enable new insights into the mechanics of the monovalent biotin/streptavidin complex under load and forced us to rethink the diversity of rupture forces reported in the literature. We discovered that the mechanical stability of this complex depends strongly on the geometry in which force is applied. By atomic force microscopy-based single molecule force spectroscopy we found unbinding of biotin to occur beyond 400 pN at force loading rates of 10 nN/s when monovalent streptavidin was tethered at its C-terminus. This value is about twice as high than that for N-terminal attachment. Steered molecular dynamics simulations provided a detailed picture of the mechanics of the unbinding process in the corresponding force loading geometries. Using machine learning techniques, we connected findings from hundreds of simulations to the experimental results, identifying different force propagation pathways. Interestingly, we observed that depending on force loading geometry, partial unfolding of N-terminal region of monovalent streptavidin occurs before biotin is released from the binding pocket.

Monodisperse measurement of the biotin-streptavidin interaction strength in a well-defined pulling geometry.

The widely used interaction of the homotetramer streptavidin with the small molecule biotin has been intensively studied by force spectroscopy and has become a model system for receptor ligand interaction. However, streptavidin's tetravalency results in diverse force propagation pathways through the different binding interfaces. This multiplicity gives rise to polydisperse force spectroscopy data. Here, we present an engineered monovalent streptavidin tetramer with a single cysteine in its functional subunit that allows for site-specific immobilization of the molecule, orthogonal to biotin binding. Functionality of streptavidin and its binding properties for biotin remain unaffected. We thus created a stable and reliable molecular anchor with a unique high-affinity binding site for biotinylated molecules or nanoparticles, which we expect to be useful for many single-molecule applications. To characterize the mechanical properties of the bond between biotin and our monovalent streptavidin, we performed force spectroscopy experiments using an atomic force microscope. We were able to conduct measurements at the single-molecule level with 1:1-stoichiometry and a well-defined geometry, in which force exclusively propagates through a single subunit of the streptavidin tetramer. For different force loading rates, we obtained narrow force distributions of the bond rupture forces ranging from 200 pN at 1,500 pN/s to 230 pN at 110,000 pN/s. The data are in very good agreement with the standard Bell-Evans model with a single potential barrier at Δx0 = 0.38 nm and a zero-force off-rate koff,0 in the 10-6 s-1 range.