Force-induced Conformational Changes Research Articles

Theoretical Aspects of the Biological Catch Bond

The biological catch bond is fascinating and counterintuitive. When an external force is applied to a catch bond, either in vivo or in vitro, the bond resists breaking and becomes stronger instead. In contrast, ordinary slip bonds, which represent the vast majority of biological and chemical bonds, dissociate faster when subjected to a force. Catch-bond behavior was first predicted theoretically 20 years ago and has recently been experimentally observed in a number of protein receptor-ligand complexes. In this Account, we review the simplest physical-chemical models that lead to analytic expressions for bond lifetime, the concise universal representations of experimental data, and the explicit requirements for catch binding. The phenomenon has many manifestations: increased lifetime with growing constant force is its defining characteristic. If force increases with time, as in jump-ramp experiments, catch binding creates an additional maximum in the probability density of bond rupture force. The new maximum occurs at smaller forces than the slip-binding maximum, merging with the latter at a certain ramp rate in a process resembling a phase transition. If force is applied periodically, as in blood flows, catch-bond properties strongly depend on force frequency. Catch binding results from a complex landscape of receptor-ligand interactions. Bond lifetime can increase if force (i) prevents dissociation through the native pathway and drives the system over a higher energy barrier or (ii) alters protein conformations in a way that strengthens receptor-ligand binding. The bond deformations can be associated with allostery; force-induced conformational changes at one end of the protein propagate to the binding site at the other end. Surrounding water creates further exciting effects. Protein-water tension provides an additional barrier that can be responsible for significant drops in bond lifetimes observed at low forces relative to zero force. This strong dependence of bond properties on weak protein-water interactions may provide universal activation mechanisms in many biological systems and create new types of catch binding. Molecular dynamics simulations provide atomistic insights: the molecular view of bond dissociation gives a foundation for theoretical models and differentiates between alternative interpretations of experimental data. The number of known catch bonds is growing; analogs are found in enzyme catalysis, peptide translocation through nanopores, DNA unwinding, photoinduced dissociation of chemical bonds, and negative thermal expansion of bulk materials, for example. Finer force resolution will likely provide many more. Understanding the properties of catch bonds offers insight into the behavior of biological systems subjected to external perturbations in general.

Single-Molecule Measurements of Force-Induced Cleavage of VWF A1A2A3-Tridomain by ADAMTS13

Binding of glycoprotein Ib (GPIb) to von Willebrand factor (VWF) mediates platelet adhesion to injured vascular surface in hemostasis, but also contributes to thrombosis under pathological high shear stress. The adhesive activity of VWF is regulated by A Disintegrin And Metalloprotease with a ThromboSpondin type 1 motifs 13 (ADAMTS13), which cleaves the ultra-large form of VWF newly secreted from activated endothelial cells to smaller and less adhesive forms. Cleavage is regulated mechanically by hemodynamic force as it occurs in the circulating blood. We used single-molecule experiments by atomic force microscopy (AFM) to test the hypothesis that mechanical stretch of VWF facilitates its proteolysis by ADAMTS13. Recombinant VWF A1A2A3 tridomain with a C-terminal histidine-tag either directly adsorbed to or captured by an anti-His monoclonal antibody (mAb) precoated on the Petri dish was stretched by either GPIb or an anti-A1 mAb (CR1) adsorbed on the AFM cantilever tip. Conformational changes, observed as abrupt length increases in the molecular complex, were induced by applied force. Times required for conformational change and for rupture of the molecular complex before or after conformational change were measured in a range of constant forces in the absence or presence of ADAMTS13. When stretched by GPIb, time-to-rupture before conformational change increased (catch) initially, reached a maximum, and then decreased (slip) with increasing force, confirming the catch-slip bond behavior we recently observed for GPIb interacting with VWF-A1 domain. Interestingly, time-to-conformational change also behaved as catch-slip bonds. Both sets of curves were independent of ADAMTS13. Time-to-rupture after conformational change exhibited the same force dependence as the time-to- rupture before conformational change. However, the time-to-rupture vs. force curve measured in the presence of ADAMTS13 was shifted downward towards shorter times relative to the curves measured in the absence of ADAMTS13. This effect was abolished by EDTA known to inhibit ADAMTS13 proteolysis. These results suggest that force-induced conformational change may have exposed the cryptic cleavage site to enable ADAMTS13 to cleave a single peptide bond in the A2 domain, thereby shortening the time-to-rupture. These data were corroborated by experiments using the CR1 mAb to stretch the A1A2A3 tridomain. The time-to-conformational change vs. force curves also exhibited catch-slip bonds, indicating that the mechanical characteristics of conformational change were indifferent to how the A1A2A3 tridomain was stretched. The time-to-rupture before conformational change vs. force curves behaved as slip bonds, consistent with the behavior of antibody-antigen interactions. Both sets of curves were independent of ADAMTS13, similar to the behavior observed when GPIb was used to stretch the A1A2A3 tridomain. Similar downward shifts were observed for the curve measured in the presence of ADAMTS13 relative to that measured in the absence of ADAMTS13. The rate of proteolytic cleavage, estimated from the downward shifts, decreased with increasing force for both cases when the A1A2A3 tridomain was stretched by GPIb and by CR1. These findings provide direct experimental evidence for mechanical force to play an important role in regulating VWF cleavage by ADAMTS13.

Visualizing myosin–actin interaction with a genetically-encoded fluorescent strain sensor

Many proteins have been shown to undergo conformational changes in response to externally applied force in vitro, but whether the force-induced protein conformational changes occur in vivo remains unclear. To reveal the force-induced conformational changes, or strains, within proteins in living cells, we have developed a genetically encoded fluorescent "strain sensor," by combining the proximity imaging (PRIM) technique, which uses spectral changes of 2 GFP molecules that are in direct contact, and myosin-actin as a model system. The developed PRIM-based strain sensor module (PriSSM) consists of the tandem fusion of a normal and circularly permuted GFP. To apply strain to PriSSM, it was inserted between 2 motor domains of Dictyostelium myosin II. In the absence of strain, the 2 GFP moieties in PriSSM are in contact, whereas when the motor domains are bound to F-actin, PriSSM has a strained conformation, leading to the loss of contact and a concomitant spectral change. Using the sensor system, we found that the position of the lever arm in the rigor state was affected by mutations within the motor domain. Moreover, the sensor was used to visualize the interaction between myosin II and F-actin in Dictyostelium cells. In normal cells, myosin was largely detached from F-actin, whereas ATP depletion or hyperosmotic stress increased the fraction of myosin bound to F-actin. The PRIM-based strain sensor may provide a general approach for studying force-induced protein conformational changes in cells.

Fragility and mechanosensing in a thermalized cytoskeleton model with forced protein unfolding

We describe a model of cytoskeletal mechanics based on the force-induced conformational change of protein cross-links in a stressed polymer network. Slow deformation of simulated networks containing cross-links that undergo repeated, serial domain unfolding leads to an unusual state-with many cross-links accumulating near the critical force for further unfolding. This state is robust to thermalization and does not occur in similar protein unbinding based simulations. Moreover, we note that the unusual configuration of near-critical protein cross-links in the fragile state provides a physical mechanism for the chemical transduction of cell-level mechanical strain and extra-cellular matrix stiffness.

Force-induced activation of Talin and its possible role in focal adhesion mechanotransduction

It is now well established that cells can sense mechanical force, but the mechanisms by which force is transduced into a biochemical signal remain poorly understood. One example is the recruitment of vinculin to reinforce initial contacts between a cell and the extracellular matrix (ECM) due to tensile force. Talin, an essential linking protein in an initial contact, contains at least one vinculin-binding site (VBS) that is cryptic and inactive in the native state. The N-terminal five-helix bundle of talin rod is a stable structure with a known cryptic VBS1. The perturbation of this stable structure through elevated temperature or destabilizing mutation activates vinculin binding. Although the disruption of this subdomain by transmitted mechanical force is a potential cue for the force-induced focal adhesion strengthening, the molecular basis for this mechanism remains elusive. Here, molecular dynamics (MD) is employed to demonstrate a force-induced conformational change that exposes the cryptic vinculin-binding residues of VBS1 to solvent under applied force along a realistic pulling direction. VBS1 undergoes a rotation of 62.0±9.5° relative to its native state as its vinculin-binding residues are released from the tight hydrophobic core. Charged and polar residues on the VBS1 surface are the site of force transmission that strongly interact with an adjacent α-helix, and in effect, apply torque to the VBS1 to cause its rotation. Activation was observed with mean force of 13.2±8.0 pN during constant velocity simulation and with steady force greater than 18.0 pN.

A Role for p130Cas in Mechanotransduction

Focal adhesions are sites of contact between cells and the extracellular matrix. Sawada et al. (2006) now report that the mechanical stretching of cells forces p130Cas, an adaptor protein at focal adhesions, to undergo a conformational change. This change promotes phosphorylation of p130Cas by Src family kinases and the transduction of integrin-mediated signaling.

Force-Induced Adsorption and Anisotropic Growth of Focal Adhesions

Focal adhesions are micrometer-sized protein aggregates that connect actin stress fibers to the extracellular matrix, a network of macromolecules surrounding tissue cells. The actin fibers are under tension due to actin-myosin contractility. Recent measurements have shown that as the actin force is increased, these adhesions grow in size and in the direction of the force. This is in contrast to the growth of condensed domains of surface-adsorbed molecules in which the dynamics are isotropic. We predict these force-sensitive, anisotropic dynamics of focal adhesions from a model for the adsorption of proteins from the cytoplasm to the adhesion site. Our theory couples the mechanical forces and elasticity to the adsorption dynamics via force-induced conformational changes of molecular-sized mechanosensors located in the focal adhesion. We predict the velocity of both the front and back of the adhesion as a function of the applied force. In addition, our results show that the relative motion of the front and back of the adhesion is asymmetric and in different ranges of forces, the adhesion can either shrink or grow in the direction of the force.

Shear-dependent 'stick-and-roll' adhesion of type 1 fimbriated Escherichia coli.

It is generally assumed that bacteria are washed off surfaces as fluid flow increases because they adhere through 'slip-bonds' that weaken under mechanical force. However, we show here that the opposite is true for Escherichia coli attachment to monomannose-coated surfaces via the type 1 fimbrial adhesive subunit, FimH. Raising the shear stress (within the physiologically relevant range) increased accumulation of type 1 fimbriated bacteria on monomannose surfaces by up to two orders of magnitude, and reducing the shear stress caused them to detach. In contrast, bacterial binding to anti-FimH antibody-coated surfaces showed essentially the opposite behaviour, detaching when the shear stress was increased. These results can be explained if FimH is force-activated; that is, that FimH mediates 'catch-bonds' with mannose that are strengthened by tensile mechanical force. As a result, on monomannose-coated surfaces, bacteria displayed a complex 'stick-and-roll' adhesion in which they tended to roll over the surface at low shear but increasingly halted to stick firmly as the shear was increased. Mutations in FimH that were predicted earlier to increase or decrease force-induced conformational changes in FimH were furthermore shown here to increase or decrease the probability that bacteria exhibited the stationary versus the rolling mode of adhesion. This 'stick-and-roll' adhesion could allow type 1 fimbriated bacteria to move along mannosylated surfaces under relatively low flow conditions and to accumulate preferentially in high shear regions.

High resolution AFM topographs of the Escherichia coli water channel aquaporin Z.

Aquaporins form a large family of membrane channels involved in osmoregulation. Electron crystallography has shown monomers to consist of six membrane spanning alpha-helices confirming sequence based predictions. Surface exposed loops are the least conserved regions, allowing differentiation of aquaporins. Atomic force microscopy was used to image the surface of aquaporin Z, the water channel of Escherichia coli. Recombinant protein with an N-terminal fragment including 10 histidines was isolated as a tetramer by Ni-affinity chromatography, and reconstituted into two-dimensional crystals with p42(1)2 symmetry. Small crystalline areas with p4 symmetry were found as well. Imaging both crystal types before and after cleavage of the N-termini allowed the cytoplasmic surface to be identified; a drastic change of the cytoplasmic surface accompanied proteolytic cleavage, while the extracellular surface morphology did not change. Flexibility mapping and volume calculations identified the longest loop at the extracellular surface. This loop exhibited a reversible force-induced conformational change.

Force-induced conformational change of bacteriorhodopsin

The cytoplasmic surface topography of purple membranes imaged by the atomic force microscope depends mainly on the force applied to the stylus. Imaged at forces of 300 pN, individual bacteriorhodopsin molecules reveal two domains. The resulting donut-shaped trimers reversibly transform into structures exhibiting three prominent protrusions when scanned at 100 pN. In parallel, the height of the protein moiety above the lipid layer increases from 4 A to 6 A. From the known structure of bacteriorhodopsin it appears that this change may be related to a bending of the most prominent cytoplasmic loop.