Single-molecule force stability of the SARS-CoV-2-ACE2 interface in variants-of-concern.

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.

Piconewton Forces Mediate GAIN Domain Dissociation of the Latrophilin-3 Adhesion GPCR.

Latrophilins are adhesion G-protein coupled receptors (aGPCRs) that control excitatory synapse formation. Most aGPCRs, including latrophilins, are autoproteolytically cleaved at their GPCR-autoproteolysis inducing (GAIN) domain, but the two resulting fragments remain noncovalently associated on the cell surface. Force-mediated dissociation of the fragments is thought to activate G-protein signaling, but how this mechanosensitivity arises is poorly understood. Here, we use magnetic tweezer assays to show that physiologically relevant forces in the 1-10 pN range lead to dissociation of the latrophilin-3 GAIN domain on the seconds-to-minutes time scale, compared to days in the absence of force. In addition, we find that the GAIN domain undergoes large changes in length in response to increasing mechanical load. These data are consistent with a model in which a force-sensitive equilibrium between compact and extended GAIN domain states precedes dissociation, suggesting a mechanism by which latrophilins and other aGPCRs may mediate mechanically induced signal transduction.

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.

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.

Different Vinculin Binding Sites Use the Same Mechanism to Regulate Directional Force Transduction.

Vinculin is a universal adaptor protein that transiently reinforces the mechanical stability of adhesion complexes. It stabilizes mechanical connections that cells establish between the actomyosin cytoskeleton and the extracellular matrix via integrins or to neighboring cells via cadherins, yet little is known regarding its mechanical design. Vinculin binding sites (VBSs) from different nonhomologous actin-binding proteins use conserved helical motifs to associate with the vinculin head domain. We studied the mechanical stability of such complexes by pulling VBS peptides derived from talin, α-actinin, and Shigella IpaA out of the vinculin head domain. Experimental data from atomic force microscopy single-molecule force spectroscopy and steered molecular dynamics (SMD) simulations both revealed greater mechanical stability of the complex for shear-like than for zipper-like pulling configurations. This suggests that reinforcement occurs along preferential force directions, thus stabilizing those cytoskeletal filament architectures that result in shear-like pulling geometries. Large force-induced conformational changes in the vinculin head domain, as well as protein-specific fine-tuning of the VBS sequence, including sequence inversion, allow for an even more nuanced force response.

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.

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.

Elastin-like Polypeptide Linkers for Single-Molecule Force Spectroscopy.

Single-molecule force spectroscopy (SMFS) is by now well established as a standard technique in biophysics and mechanobiology. In recent years, the technique has benefitted greatly from new approaches to bioconjugation of proteins to surfaces. Indeed, optimized immobilization strategies for biomolecules and refined purification schemes are being steadily adapted and improved, which in turn has enhanced data quality. In many previously reported SMFS studies, poly(ethylene glycol) (PEG) was used to anchor molecules of interest to surfaces and/or cantilever tips. The limitation, however, is that PEG exhibits a well-known trans-trans-gauche to all-trans transition, which results in marked deviation from standard polymer elasticity models such as the worm-like chain, particularly at elevated forces. As a result, the assignment of unfolding events to protein domains based on their corresponding amino acid chain lengths is significantly obscured. Here, we provide a solution to this problem by implementing unstructured elastin-like polypeptides as linkers to replace PEG. We investigate the suitability of tailored elastin-like polypeptides linkers and perform direct comparisons to PEG, focusing on attributes that are critical for single-molecule force experiments such as linker length, monodispersity, and bioorthogonal conjugation tags. Our results demonstrate that by avoiding the ambiguous elastic response of mixed PEG/peptide systems and instead building the molecular mechanical systems with only a single bond type with uniform elastic properties, we improve data quality and facilitate data analysis and interpretation in force spectroscopy experiments. The use of all-peptide linkers allows alternative approaches for precisely defining elastic properties of proteins linked to surfaces.

Monovalent Strep-Tactin for strong and site-specific tethering in nanospectroscopy.

Strep-Tactin, an engineered form of streptavidin, binds avidly to the genetically encoded peptide Strep-tag II in a manner comparable to streptavidin binding to biotin. These interactions have been used in protein purification and detection applications. However, in single-molecule studies, for example using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), the tetravalency of these systems impedes the measurement of monodispersed data. Here, we introduce a monovalent form of Strep-Tactin that harbours a unique binding site for Strep-tag II and a single cysteine that allows Strep-Tactin to specifically attach to the atomic force microscope cantilever and form a consistent pulling geometry to obtain homogeneous rupture data. Using AFM-SMFS, the mechanical properties of the interaction between Strep-tag II and monovalent Strep-Tactin were characterized. Rupture forces comparable to biotin:streptavidin unbinding were observed. Using titin kinase and green fluorescent protein, we show that monovalent Strep-Tactin is generally applicable to protein unfolding experiments. We expect monovalent Strep-Tactin to be a reliable anchoring tool for a range of single-molecule studies.