Catch Bonds Research Articles

A Simple Mechanical Model for Synthetic Catch Bonds

Summary Catch bonds are protein-ligand bonds that become more difficult to break as the applied force increases, a counterintuitive phenomenon that has not yet been reproduced in synthetic systems. Here, we have demonstrated that a simple mechanical design based on a tweezer-like mechanism can exhibit catch bond characteristics under thermal excitations. The tweezer has a force-sensitive switch that controls the transition of the system to a high-ligand-affinity state with additional ligand-tweezer interactions. Applying kinetic theory to a two-mass-two-spring idealized model of the tweezer, we show that by tuning the shape of the switch and the ligand-tweezer interaction energy landscapes, we can achieve greater lifetimes at larger force levels. We validate our theory with molecular dynamics simulations and produce a characteristic lifetime curve reminiscent of catch bonds. Our analysis reveals minimal design guidelines for reproducing the catch bond phenomenon in synthetic systems such as molecular switches/foldamers, DNA linkers, and nanoparticle networks.

Force-Dependent Facilitated Dissociation Can Generate Protein-DNA Catch Bonds

Cellular structures are continually subjected to forces, which may serve as mechanical signals for cells through their effects on biomolecule interaction kinetics. Typically, molecular complexes interact via “slip bonds,” so applied forces accelerate off rates by reducing transition energy barriers. However, biomolecules with multiple dissociation pathways may have considerably more complicated force dependencies. This is the case for DNA-binding proteins that undergo “facilitated dissociation,” in which competitor biomolecules from solution enhance molecular dissociation in a concentration-dependent manner. Using simulations and theory, we develop a generic model that shows that proteins undergoing facilitated dissociation can form an alternative type of molecular bond, known as a “catch bond,” for which applied forces suppress protein dissociation. This occurs because the binding by protein competitors responsible for the facilitated dissociation pathway can be inhibited by applied forces. Within the model, we explore how the force dependence of dissociation is regulated by intrinsic factors, including molecular sensitivity to force and binding geometry and the extrinsic factor of competitor protein concentration. We find that catch bonds generically emerge when the force dependence of the facilitated unbinding pathway is stronger than that of the spontaneous unbinding pathway. The sharpness of the transition between slip- and catch-bond kinetics depends on the degree to which the protein bends its DNA substrate. This force-dependent kinetics is broadly regulated by the concentration of competitor biomolecules in solution. Thus, the observed catch bond is mechanistically distinct from other known physiological catch bonds because it requires an extrinsic factor—competitor proteins—rather than a specific intrinsic molecular structure. We hypothesize that this mechanism for regulating force-dependent protein dissociation may be used by cells to modulate protein exchange, regulate transcription, and facilitate diffusive search processes.

TCR–pMHC kinetics under force in a cell-free system show no intrinsic catch bond, but a minimal encounter duration before binding

The T cell receptor (TCR)-peptide-MHC (pMHC) interaction is the only antigen-specific interaction during T lymphocyte activation. Recent work suggests that formation of catch bonds is characteristic of activating TCR-pMHC interactions. However, whether this binding behavior is an intrinsic feature of the molecular bond, or a consequence of more complex multimolecular or cellular responses, remains unclear. We used a laminar flow chamber to measure, first, 2D TCR-pMHC dissociation kinetics of peptides of various activating potency in a cell-free system in the force range (6 to 15 pN) previously associated with catch-slip transitions and, second, 2D TCR-pMHC association kinetics, for which the method is well suited. We did not observe catch bonds in dissociation, and the off-rate measured in the 6- to 15-pN range correlated well with activation potency, suggesting that formation of catch bonds is not an intrinsic feature of the TCR-pMHC interaction. The association kinetics were better explained by a model with a minimal encounter duration rather than a standard on-rate constant, suggesting that membrane fluidity and dynamics may strongly influence bond formation.

Pilus-1 Backbone Protein RrgB of Streptococcus pneumoniae Binds Collagen I in a Force-Dependent Way.

Attachment to host tissue is a prerequisite for successful host colonization and invasion of pathogens. Many pathogenic bacteria use surface appendices, called pili, to bind and firmly attach to host tissue surfaces. Although it has been speculated that the laterally positioned D3 domain of the pilus-1 backbone protein RrgB of Streptococcus pneumoniae may promote bacterial-host interaction, via adhesion to extracellular matrix molecules, such as collagen, earlier studies showed no affinity of RrgB to collagen I. Using atomic force microscopy-based single molecule force spectroscopy combined with lateral force microscopy, we show that under mechanical load, RrgB in fact binds to human collagen I in a force-dependent manner. We observe exceptionally strong interactions, with interaction forces reaching as much as 1500 pN, and we show that high force loading and shearing rates enhance and further strengthen the interaction. In addition, the affinity of RrgB to collagen I under mechanical load not only depends on the orientation of the D3 domain but also on the orientation of the collagen fibrils, relative to the pulling direction. Both exceptionally high binding forces and force-induced bond strengthening resemble the behavior of so-called catch bonds, which have recently been observed in bacterial adhesins, but have not been reported for multimeric backbone subunits of virulence related pili.

Mechanical feedback enables catch bonds to selectively stabilize scanning microvilli at T-cell surfaces.

T-cells use microvilli to search the surfaces of antigen-presenting cells for antigenic ligands. The active motion of scanning microvilli provides a force-generating mechanism that is intriguing in light of single-molecule experiments showing that applied forces increase the lifetimes of stimulatory receptor–ligand bonds (catch-bond behavior). In this work, we introduce a theoretical framework to explore the motion of a microvillar tip above an antigen-presenting surface when receptors on the tip stochastically bind to ligands on the surface and dissociate from them in a force-dependent manner. Forces on receptor-ligand bonds impact the motion of the microvillus, leading to feedback between binding and microvillar motion. We use computer simulations to show that the average microvillar velocity varies in a ligand-dependent manner; that catch bonds generate responses in which some microvilli almost completely stop, while others move with a broad distribution of velocities; and that the frequency of stopping depends on the concentration of stimulatory ligands. Typically, a small number of catch bonds initially immobilize the microvillus, after which additional bonds accumulate and increase the cumulative receptor-engagement time. Our results demonstrate that catch bonds can selectively slow and stabilize scanning microvilli, suggesting a physical mechanism that may contribute to antigen discrimination by T-cells.

Force-Dependent Unbinding Rate of Molecular Motors from Stationary Optical Trap Data.

Molecular motors walk along filaments until they detach stochastically with a force-dependent unbinding rate. Here, we show how this unbinding rate can be obtained from the analysis of experimental data of molecular motors moving in stationary optical traps. Two complementary methods are presented, based on the analysis of the distribution for the unbinding forces and of the motor's force traces. In the first method, analytically derived force distributions for slip bonds, slip-ideal bonds, and catch bonds are used to fit the cumulative distributions of the unbinding forces. The second method is based on the statistical analysis of the observed force traces. We validate both methods with stochastic simulations and apply them to experimental data for kinesin-1.

Conformational Dynamics of T-Cell Receptor Chassis for Mechanosensing

AlphaBeta T-cell receptors (TCRαβ) are the major components of the adaptive immune system, sensing foreign antigens displayed on the surface of mammalian host cells altered by infection or other transformations with a remarkably high signal to noise ratio. Despite their high sensitivity, the affinity of each to the antigen peptide-bound major histocompatibility complex (pMHC) is low in thermal equilibrium, typically in the micromolar range. An emerging concept that reconciles the conundrum of high sensitivity versus low affinity is that the TCRαβ functions as a mechanosensor. Mechanical load on the TCRαβ-pMHC complex enhances the bond lifetime via activation of a catch bond pathway, a mechanism which is largely uncharacterized in structural terms. Past studies focused on behaviors of contacts directly involving TCRαβ and pMHC surfaces. However, such localized interactions alone cannot account for the catch bond since there is an allostery that modulates the catch bond behavior through TCR subdomains distal to the TCRαβ-pMHC interface. Thus, understanding of the conformational behavior of the entire TCR protein is required. Here we perform atomistic molecular dynamics simulations of TCRαβ as well as TCR gamma-delta (TCRγδ), receptors of cognate and innate T-cell immunity subsets, respectively. We also study a TCRγδ-αβ chimera that has the antigen-binding variable domains of TCRγδ with the membrane-proximal TCRαβ constant domains. By building ball-and-stick models of TCRs based on simulation trajectories, we examine relative motions of the four subdomains of each TCR type. We find that interdomain motion affects the organization of the pMHC-binding complementarity determining loops. These results suggest a TCR catch bond mechanism whereby an applied load alters the conformational dynamics of the subdomains and in turn, controls the TCR-pMHC interaction including resultant bond lifetime.

Mechanical Strength of Catch Bond Forming FimH and Mannose

Adhesive interactions must withstand mechanical forces occurring in an organism. Forces have influenced some proteins such as integrins, clotting factors, and bacterial adhesive proteins to develop catch bond properties, in which the lifetime between a biomolecule and ligand increases with applied force. One of the most well studied catch bond behaviors is found between the E. coli adhesive protein, FimH, and its ligand, mannose. FimH has two distinct conformations with significantly different mannose affinities. The low affinity conformation predominates the initial FimH-mannose interactions, as this state, characterized with an open binding pocket, facilitates fast kinetics. A force pulling on the FimH-mannose interaction elongates FimH, inducing a transition into the high mannose affinity state, which includes a closed mannose binding pocket. While in this force-stabilized high affinity conformation, FimH has a remarkably long-lived lifetime that has never been fully measured before. We suspect the closed binding pocket is responsible for this lifetime, and mannose dissociation occurs when the pocket spontaneously opens. If the opening of this pocket occurs independent of the strength of the applied force, mannose unbinding would thus represent a force independent, or ideal bond, interaction. To explore this possibility, we measured the lifetimes of the mannose-FimH interaction at various forces with FimH in the high affinity state. We utilized a magnetic tweezer apparatus, to apply various constant forces to mannose-coated magnetic beads bound to immobilized FimH. While true ideal bond behavior cannot be concluded with certainty, the FimH-mannose bond often outlasted the 15-minute collection period, even at high forces relative to other catch bond forming proteins. Understanding the relationship between force and FimH's incredibly stable binding pocket, would grant further insight into the catch bond mechanism associated with FimH.

Exploring the Sulfatase 1 Catch Bond Free Energy Landscape using Jarzynski's Equality

In non-covalent biological adhesion, molecular bonds commonly exhibit a monotonously decreasing lifetime when subjected to tensile forces (slip bonds). In contrast, catch bonds behave counter intuitively, as they show an increased lifetime within a certain force interval. To date only a hand full of catch bond displaying systems have been identified. In order to unveil their nature, a number of structural and phenomenological models have been introduced. Regardless of the individual causes for catch bond behavior, it appears evident that the free energy landscapes of these interactions bear more than one binding state. Here, we investigated the catch bond interaction between the hydrophilic domain of the human cell surface sulfatase 1 (Sulf1HD) and its physiological substrate heparan sulfate (HS) by atomic force microscopy based single molecule force spectroscopy (AFM-SMFS). Using Jarzynski's equality, we estimated the associated Gibbs free energy and provide a comprehensive thermodynamic and kinetic characterization of Sulf1HD/HS interaction. Interestingly, the binding potential landscape exhibits two distinct potential wells, which confirms the recently suggested two state binding. Even though structural data of Sulf1HD is lacking, our results allow drafting a detailed picture of the directed and processive desulfation of HS.

A Simple Mechanical Model for Synthetic Catch Bonds

Catch bonds are protein-ligand bonds which become more difficult to break with larger applied force, a counterintuitive phenomenon that is yet to be reproduced in synthetic systems. Here, we have demonstrated that a simple mechanical design based on a tweezer-like mechanism can exhibit catch bond characteristics under thermal excitations. The tweezer has a force-sensitive switch which controls the transition of the system to a high-ligand-affinity state with additional ligand-tweezer interactions. Applying kinetic theory to a two-mass-two-spring idealized model of the tweezer, we show that by tuning the shape of the switch and the ligand-tweezer interaction energy landscapes, we can achieve greater lifetimes at larger force levels. We validate our theory with molecular dynamics simulations and produce a characteristic lifetime curve reminiscent of catch bonds. Our analysis reveals minimal design guidelines for reproducing the catch bond phenomenon in synthetic systems such as molecular switches/foldamers, DNA linkers and nanoparticle networks.

Molecular Mechanisms of Transition from Catch to Slip Bonds in Fibrin

Fibrin is the end product of blood clotting that constitutes a proteinaceous three dimensional network providing the filamentous mechanical scaffold of clots and thrombi. Formation of fibrin is essential for stopping bleeding (hemostasis) and thrombotic obstruction of blood vessels (thrombosis). In order to explore how mechanical load impacts fibrin, we carried out optical trap-based single-molecule forced unbinding assays. Surprisingly, the strength of non-covalent A:a knob-hole interactions, most important in fibrin polymerization, first increases with tensile force (catch bonds) and then decreases when the force exceeds a critical value (slip bonds). To provide the structural basis of the catch-slip bond transition, we performed molecular modeling of A:a knob-hole complex. The results revealed that the key structural element, a movable flap (residues γ295-γ305), serves as a tension-dependent sensor. Flap dissociation from the regulatory B-domain in the γ-nodule and subsequent translocation to knob ‘A’ triggers the hole ‘a’ closure resulting in the force-dependent increase of binding affinity and prolonged bond lifetimes. The experimental discovery of bi-phasic catch-slip kinetics of knob-hole bond rupture, supported by simulations, is quantitatively explained using a theory, formulated in terms of dynamic remodeling of the binding interface fluctuating between the low (slip) and high (catch) affinity states. We estimated the stiffness of the binding interface to be 15.7 pN/nm and the width of interface fluctuations to be 0.7-2.7 nm. Strengthening of the A:a knob-hole bonds at small (<30-40 pN) forces might favor formation and reinforcement of nascent fibrin clots subject to hydrodynamic shear in vivo. The novel and unexplored mechano-chemical aspects of fibrin polymerization addressed in this study for the first time further advances our understanding of blood clotting. The work was supported, in part, by the Program for Competitive Growth at Kazan Federal University.

Mechano-regulation of Peptide-MHC Class I Conformations Determines TCR Antigen Recognition

TCRs recognize cognate pMHCs to initiate Tcell signaling and adaptive immunity. Mechanical force strengthens TCR-pMHC interactions to elicit agonist-specific catch bonds to trigger TCR signaling, but the underlying dynamic structural mechanism is unclear. We combined steered molecular dynamics (SMD) simulation, single-molecule biophysical approaches, and functional assays to collectively demonstrate that mechanical force induces conformational changes in pMHCs to enhance pre-existing contacts and activates new interactions at the TCR-pMHC binding interface to resist bond dissociation under force, resulting in TCR-pMHC catch bondsand Tcell activation. Intriguingly, cancer-associatedsomatic mutations in HLA-A2 that may restrict these conformational changes suppressed TCR-pMHC catch bonds. Structural analysis also indicated that HLA polymorphism might alter the equilibrium of these conformational changes. Our findings not only reveal critical roles of force-induced conformational changes in pMHCs for activating TCR-pMHC catch bonds but also have implications for Tcell-based immunotherapy.

Stochastic modeling reveals how motor protein and filament properties affect intermediate filament transport

Intermediate filaments are a key component of the cytoskeleton. Their transport along microtubules plays an essential role in the control of the shape and structural organization of cells. To identify the key parameters responsible for the control of intermediate filament transport, we generated a model of elastic filament transport by microtubule-associated dynein and kinesin. The model is also applicable to the transport of any elastically-coupled cargoes. We investigate the effect of filament properties such as number of motor binding sites, length, and elasticity on motion of filaments. Additionally, we consider the effect of motor properties, i.e. off rates, on filament transport. When one motor has a catch bond off rate it dictates the motion, whereas when motors have the same type of off rate filaments can alternate between retrograde and anterograde motions. The elasticity of filaments optimizes the filament transport and the coordination of motors along the length of the filament.

Exploring the Sulfatase 1 Catch Bond Free Energy Landscape using Jarzynski\u2019s Equality

In non-covalent biological adhesion, molecular bonds commonly exhibit a monotonously decreasing life time when subjected to tensile forces (slip bonds). In contrast, catch bonds behave counter intuitively, as they show an increased life time within a certain force interval. To date only a hand full of catch bond displaying systems have been identified. In order to unveil their nature, a number of structural and phenomenological models have been introduced. Regardless of the individual causes for catch bond behavior, it appears evident that the free energy landscapes of these interactions bear more than one binding state. Here, we investigated the catch bond interaction between the hydrophilic domain of the human cell surface sulfatase 1 (Sulf1HD) and its physiological substrate heparan sulfate (HS) by atomic force microscopy based single molecule force spectroscopy (AFM-SMFS). Using Jarzynski’s equality, we estimated the associated Gibbs free energy and provide a comprehensive thermodynamic and kinetic characterization of Sulf1HD/HS interaction. Interestingly, the binding potential landscape exhibits two distinct potential wells which confirms the recently suggested two state binding. Even though structural data of Sulf1HD is lacking, our results allow to draft a detailed picture of the directed and processive desulfation of HS.

A TCR mechanotransduction signaling loop induces negative selection in the thymus.

The T cell antigen receptor (TCR) expressed on thymocytes interacts with self-peptide major histocompatibility complex (pMHC) ligands to signal apoptosis or survival. Here, we found that negative-selection ligands induced thymocytes to exert forces on the TCR and the co-receptor CD8 and formed cooperative TCR-pMHC-CD8 trimolecular 'catch bonds', whereas positive-selection ligands induced less sustained thymocyte forces on TCR and CD8 and formed shorter-lived, independent TCR-pMHC and pMHC-CD8 bimolecular 'slip bonds'. Catch bonds were not intrinsic to either the TCR-pMHC or the pMHC-CD8 arm of the trans (cross-junctional) heterodimer but resulted from coupling of the extracellular pMHC-CD8 interaction to the intracellular interaction of CD8 with TCR-CD3 via associated kinases to form a cis (lateral) heterodimer capable of inside-out signaling. We suggest that the coupled trans-cis heterodimeric interactions form a mechanotransduction loop that reinforces negative-selection signaling that is distinct from positive-selection signaling in the thymus.

Unraveling the mechanism of the cadherin-catenin-actin catch bond.

The adherens junctions between epithelial cells involve a protein complex formed by E-cadherin, β-catenin, α-catenin and F-actin. The stability of this complex was a puzzle for many years, since in vitro studies could reconstitute various stable subsets of the individual proteins, but never the entirety. The missing ingredient turned out to be mechanical tension: a recent experiment that applied physiological forces to the complex with an optical tweezer dramatically increased its lifetime, a phenomenon known as catch bonding. However, in the absence of a crystal structure for the full complex, the microscopic details of the catch bond mechanism remain mysterious. Building on structural clues that point to α-catenin as the force transducer, we present a quantitative theoretical model for how the catch bond arises, fully accounting for the experimental lifetime distributions. The underlying hypothesis is that force induces a rotational transition between two conformations of α-catenin, overcoming a significant energy barrier due to a network of salt bridges. This transition allosterically regulates the energies at the interface between α-catenin and F-actin. The model allows us to predict these energetic changes, as well as highlighting the importance of the salt bridge rotational barrier. By stabilizing one of the α-catenin states, this barrier could play a role in how the complex responds to additional in vivo binding partners like vinculin. Since significant conformational energy barriers are a common feature of other adhesion systems that exhibit catch bonds, our model can be adapted into a general theoretical framework for integrating structure and function in a variety of force-regulated protein complexes.

Adhesion of Staphylococcus aureus to Corneocytes from Atopic Dermatitis Patients Is Controlled by Natural Moisturizing Factor Levels.

ABSTRACTThe bacterial pathogen Staphylococcus aureus plays an important role in atopic dermatitis (AD), a chronic disorder that mostly affects children. Colonization of the skin of AD patients by S. aureus exacerbates the disease, but the molecular determinants of the bacterium-skin adhesive interactions are poorly understood. Specifically, reduced levels of natural moisturizing factor (NMF) in the stratum corneum have been shown to be associated with more severe AD symptoms, but whether this is directly related to S. aureus adhesion is still an open question. Here, we demonstrate a novel relationship between NMF expression in AD skin and strength of bacterial adhesion. Low-NMF corneocytes, unlike high-NMF ones, are covered by a dense layer of nanoscale villus protrusions. S. aureus bacteria isolated from AD skin bind much more strongly to corneocytes when the NMF level is reduced. Strong binding forces originate from a specific interaction between the bacterial adhesion clumping factor B (ClfB) and skin ligands. Remarkably, mechanical tension dramatically strengthens ClfB-mediated adhesion, as observed with catch bonds, demonstrating that physical stress plays a role in promoting colonization of AD skin by S. aureus. Collectively, our findings demonstrate that patient NMF levels regulate the strength of S. aureus-corneocyte adhesion, the first step in skin colonization, and suggest that the ClfB binding mechanism could represent a potential target for new therapeutic treatments.

Regulatory element in fibrin triggers tension-activated transition from catch to slip bonds

Fibrin formation and mechanical stability are essential in thrombosis and hemostasis. To reveal how mechanical load impacts fibrin, we carried out optical trap-based single-molecule forced unbinding experiments. The strength of noncovalent A:a knob-hole bond stabilizing fibrin polymers first increases with tensile force (catch bonds) and then decreases with force when the force exceeds a critical value (slip bonds). To provide the structural basis of catch-slip-bond behavior, we analyzed crystal structures and performed molecular modeling of A:a knob-hole complex. The movable flap (residues γ295 to γ305) containing the weak calcium-binding site γ2 serves as a tension sensor. Flap dissociation from the B domain in the γ-nodule and translocation to knob 'A' triggers hole 'a' closure, resulting in the increase of binding affinity and prolonged bond lifetimes. The discovery of biphasic kinetics of knob-hole bond rupture is quantitatively explained by using a theory, formulated in terms of structural transitions in the binding pocket between the low-affinity (slip) and high-affinity (catch) states. We provide a general framework to understand the mechanical response of protein pairs capable of tension-induced remodeling of their association interface. Strengthening of the A:a knob-hole bonds at 30- to 40-pN forces might favor formation of nascent fibrin clots subject to hydrodynamic shear in vivo.

Isolation of a Structural Mechanism for Uncoupling T Cell Receptor Signaling from Peptide-MHC Binding

TCR-signaling strength generally correlates with peptide-MHC binding affinity; however, exceptions exist. We find high-affinity, yet non-stimulatory, interactions occur with high frequency in the human Tcell repertoire. Here, we studied human TCRs that are refractory to activation by pMHC ligands despite robust binding. Analysis of 3D affinity, 2D dwell time, and crystal structures of stimulatory versus non-stimulatory TCR-pMHC interactions failed to account for their different signaling outcomes. Using yeast pMHC display, we identified peptide agonists of a formerly non-responsive TCR. Single-molecule force measurements demonstrated the emergence of catch bonds in the activating TCR-pMHC interactions, correlating with exclusion of CD45 from the TCR-APC contact site. Molecular dynamics simulations of TCR-pMHC disengagement distinguished agonist from non-agonist ligands based on the acquisition of catch bonds withinthe TCR-pMHC interface. The isolation of catch bonds as a parameter mediating the coupling of TCR binding and signaling has important implications for TCR and antigen engineering for immunotherapy.

Molecular force spectroscopy of kinetochore-microtubule attachment in silico: Mechanical signatures of an unusual catch bond and collective effects.

Measurement of the lifetime of attachments formed by a single microtubule (MT) with a single kinetochore (kt) in vitro under force-clamp conditions had earlier revealed a catch-bond-like behavior. In the past, the physical origin of this apparently counterintuitive phenomenon was traced to the nature of the force dependence of the (de)polymerization kinetics of the MTs. Here, first the same model MT-kt attachment is subjected to external tension that increases linearly with time until rupture occurs. In our force-ramp experiments in silico, the model displays the well known "mechanical signatures" of a catch bond probed by molecular force spectroscopy. Exploiting this evidence, we have further strengthened the analogy between MT-kt attachments and common ligand-receptor bonds in spite of the crucial differences in their underlying physical mechanisms. We then extend the formalism to model the stochastic kinetics of an attachment formed by a bundle of multiple parallel microtubules with a single kt considering the effect of rebinding under force-clamp and force-ramp conditions. From numerical studies of the model we predict the trends of variation of the mean lifetime and mean rupture force with the increasing number of MTs in the bundle. Both the mean lifetime and the mean rupture force display nontrivial nonlinear dependence on the maximum number of MTs that can attach simultaneously to the same kt.