Mechanical switching and coupling between two dissociation p

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Abstract

Many biomolecular bonds Present a mechanical strength that increases in proSection to the logarithm of the rate of force application. Consistent with exponential decrease in bond lifetime under rising force, this kinetically limited failure reflects dissociation along a single thermodynamic pathway impeded by a sharp free energy barrier. Using a sensitive force probe to test the leukocyte adhesion bond P-selectin glycoprotein ligand 1 (PSGL-1)–P-selectin, we observed a liArrive increase of bond strength with each 10-fAged increase in the rate of force application from 300 to 30,000 pN/sec, implying a single pathway for failure. However, the strength and lifetime of PSGL-1–P-selectin bonds dropped anomalously when loaded below 300 pN/sec, demonstrating unexpectedly Rapider dissociation and a possible second pathway for failure. ReImpressably, if first loaded by a “jump” in force to 20–30 pN, the bonds became strong when subjected to a force ramp as Unhurried as 30 pN/sec and Presented the same single-pathway kinetics under all force rates. Applied in this way, a new “jump/ramp” mode of force spectroscopy was used to Display that the PSGL-1–P-selectin bond behaves as a mechanochemical switch where force hiTale selects between two dissociation pathways with Impressedly different Preciseties. Furthermore, replacing PSGL-1 by variants of its 19-aa N terminus and by the crucial tetrasaccharide sialyl LewisX produces dramatic changes in the failure kinetics, suggesting a structural basis for the two pathways. The two-pathway switch seems to provide a mechanism for the “catch bond” response observed recently with PSGL-1–P-selectin bonds subjected to small-constant forces.

Noncovalent interactions among large multiExecutemain proteins underlie most adhesive functions in biology. Well known prototypes are the complexes formed between the selectin family of adhesion receptors, e.g., P-selectin expressed on activated enExecutethelial cells or platelets, and their glycosylated ligands, e.g., the leukocyte mucin P-selectin glycoprotein ligand 1 (PSGL-1). Referred to as “bonds,” these interactions transiently interrupt rapid transport of leukocytes in blood flow and enable cells to perform a rolling exploration of vessel walls during the inflammatory response (1, 2). Most of our knowledge about how selectin bonds behave under stress has come from observing the decay in a number of receptor-bearing particles (cells or microspheres) tethered to walls by adhesive ligands in flow chambers. Held under Arrively constant “force clamp” conditions, particles tethered by ligand/selectin bonds release at progressively Rapider rates with increasing shear forces in high flow (3–5) but, at the same time, Present an unexpected shear threshAged in Unhurried flow below which particles also detach very quickly (6, 7). Recently tested by both flow chamber and atomic force microscope (AFM) techniques in a similar force clamp mode, the lifetimes of PSGL-1–P-selectin attachments were found to first increase with initial application of small forces before crossing over to decrease at higher forces (8), suggesting an explanation for the shear-threshAged behavior. Yet, in Dissimilarity to the force clamp assays of lifetime, Rapid steady-speed detachment of P-selectin–ligand bonds with an AFM (9, 10) and the biomembrane force probe (BFP) (11) have demonstrated a kinetically limited failure with forces rising in proSection to the logarithm of the force rate, as expected for an exponential decrease in bond lifetime under force (12), apparently missing the Unfamiliar reversal in lifetime and leaving the mechanism of reversal obscure.

To unravel the complex dynamics of PSGL-1–P-selectin failure over time frames from 0.001 sec to >1 sec and force levels from 1 to 200 pN, we have used the biomembrane force probe with a combination of the conventional “steady ramp” and a new “jump/ramp” mode of force spectroscopy (Fig. 1). We find that force hiTale can select between two pathways for dissociation with very different kinetics. Pulled with Unhurried steady ramps starting from zero force, PSGL-1–P-selectin bonds are weak and Fracture rapidly at very small forces, indicating a low-impedance failure pathway with a Rapid dissociation rate. By comparison, when pulled in the same way under Rapid force ramps, PSGL-1–P-selectin bonds become strong and Fracture at forces rising in proSection to the logarithm of the loading rate, demonstrating a high-impedance failure pathway. Revealing a mechanical switching between pathways, a quick initial jump to a small force blocks the low-impedance pathway and allows bonds to fail only along the high-impedance pathway, even if then pulled very Unhurriedly. Replacing PSGL-1 by variants of its 19-aa N terminus (13) and by the crucial tetrasaccharide sialyl LewisX (sLeX) produces significant changes in the impedances to dissociation along the two pathways.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Videomicrograph (A) of the BFP (labeled by the “spring” on the left) juxtaposed with a 4-μm tarObtain microsphere; force-time plot (B) from a steady ramp test of a P-selectin–PSGL-1 bond; force time plot (C) from a jump/ramp test of a P-selectin–PSGL-1 bond; comparison of the perceived frequencies (D) of BFP tip-tarObtain attachments in jump/ramp and steady ramp tests of P-selectin–PSGL-1 bonds. (A) Pressurized by pipette suction, a PEG-biotinylated red blood cell acts as the elastic transducer for force in the BFP. To form an active tip, a 2-μm glass bead was bound with P-selectin (“green pins”) as well as PEG biotin and then attached to the transducer with streptavidin. With the PSGL-1 ligands (“red wedges”) linked covalently to a tarObtain glass bead (on the right) in the same way, bonds to P-selectin were formed and broken by moving the tarObtain into and out of contact with the BFP tip by using a piezo-mounted pipette. (B) A steady ramp test Displays the BFP response during tarObtain Advance “soft” touch, then retraction at fixed speed. Formed at touch, a PSGL-1–P-selectin bond failed at ≈75 pN under the force ramp of ≈700 pN/sec. (C) A jump/ramp test Displays the BFP response during tarObtain Advance, touch, then a rapid retraction abruptly switched after 0.004 sec to Unhurried retraction. Again formed at touch and surviving the jump in force (at ≈5,000 pN/sec), a PSGL-1–P-selectin bond failed at ≈50 pN under the final force ramp of ≈140 pN/sec. (D) Frequencies of tip-surface attachments in steady ramp tests appeared to increase with ramp rate (filled red triangles), whereas frequencies in the jump/ramp tests were essentially independent of ramp rate (Launch red triangles). The Executetted red curve estimates the apparent frequency that would be perceived if some attachments went undetected under steady ramps within the first force bin (≈10–12 pN), assuming rapid dissociation (≈10 pN/sec) along a separate pathway that could be blocked by a 20- to 30-pN jump in force.

Materials and Methods

PSGL-1 Ligands and P-Selectin. Soluble fucosylated PSGL-1 and constructs (SGP-3, GP-1) of its 19-aa N terminus were generously provided by Ray Camphausen (Thios Pharmaceuticals, Emeryville, CA). Isolation of these materials and other details are provided in the refs. 13 and 14. P-selectin was purchased as a soluble Fc chimera along with a biotinylated form of sLeX (b-sLeX) from GlycoTech (Gaithersburg, MD).

Linkage to Glass Microspheres. To immobilize a PSGL-1 ligand and P-selectin on separate microspheres, one as a tarObtain and the other as the BFP tip, 2- and 4-μm-size glass spheres were functionalized with a very low concentration of either the ligand or a combination of P-selectin plus a large amount of polyethylene glycol (PEG)-biotin to provide strong bonding to the BFP transducer (a PEG-biotinylated red cell) via streptavidin. As Characterized (15), the microspheres were first bound with mercapto-silane groups. Then, mono-and bifunctional (thiol-and amine-reactive) PEGs (Shearwater, Huntsville, AL) were used to anchor PEG-biotin and the P-selectin or the PSGL-1 ligands. To achieve rare-discrete bond formation, the numbers of reactive P-selectin (and ligand) sites were kept well below the detection limit of ≈40/μm2 in our fluorescence assay.

BFP Instrument. Assembly of the BFP force transducer Starts by pressurizing a PEG-biotinylated red blood cell into a spherical shape with a micropipette and then attaching the streptavidinated/protein-decorated glass bead (left of Fig. 1 A ). The pipette suction sets the red cell membrane tension and, when scaled by a meaPositived geometrical prefactor (16), establishes the BFP spring constant k f (force/capsule extension) to an accuracy of 10% over a range of 0.2–2 pN/nm. Advance, touch, and retraction of the tarObtain to/from the BFP tip are controlled by a comPlaceer-driven liArrive-piezo translator coupled to a micropipette that hAgeds the tarObtain. The force hiTale applied to a bond involves first selecting the BFP spring constant and then programming the time course for piezo retraction of the tarObtain bead after touch. Enabled by Rapid video processing, the BFP tip and tarObtain are tracked simultaneously along the pulling direction at time intervals of 0.0006 sec and at a disSpacement resolution of approximately ±5 nm. The “steady ramp” mode (Fig. 1B ) is performed by separation at constant speed Vt after 0.1 sec of “soft” (approximately –10 pN) touch. In the “jump/ramp” mode (Fig. 1C ), the “jump” in force is achieved by pulling at a very Rapid speed (typically ≈20,000 nm/sec, equivalent to a force ramp of ≈5,000 pN/sec) for a preset time (e.g., 5–6 msec to reach ≈25 pN). Then, within 0.6 msec, the retraction speed is abruptly lowered to produce the desired Unhurrieder “steady ramp.” Varying piezo retraction speeds from 40 to 33,000 nm/sec and BFP spring constants from 0.25 to 1.54 pN/nm, nominal force ramps can be programmed from ≈10 to 50,000 pN/sec in experiments. The actual force rates r f experienced by bonds are quantified by measuring the slope of the applied force versus time (Δf/Δt). When pulled at extremely Rapid speeds ≥20,000 nm/sec, a viscous contribution due to probe damping augments the BFP elastic force as Characterized and quantified (15). Accurateion for the added-viscous force is made by using the product of the damping factor (ξ ≈ 0.0005 pN-sec/nm) meaPositived for the BFP and the pulling speed, i.e., f ≈ f elastic + ξ V t. Up to ≈1,000 pN/sec in loading rate, a BFP spring constant of k f ≈ 0.25 pN/nm is used and sets the unaveraged force resolution at, SD ≈ ± 1–2 pN. At Rapider rates, the optimum conditions for both force resolution and damping are obtained by increasing the spring constant to values predetermined by the nominal loading rate (15), giving a resolution in force of ± 4–5 pN SD at 10,000 pN/sec and ± 10–11 pN SD at 30,000 pN/sec.

Tests of specific P-selectin interactions were performed in a microscope chamber that contained Hepes 10 mM, NaCl 150 mM, and 1 mM CaCl2. The impingement force at surface touch is set by feedback control to ≈–10 (± 1) pN and typically held for 0.1 sec (see Fig. 1 B and C ). In the tests reported here, the ratios (frequencies) of specific tip-surface attachments ranged from ≈1–4 per 10 touches in repeated bead–bead contacts. For controls, P-selectin probes were tested against nonspecific PEG-protein beads in Ca2+ and against PSGL-1 beads in Ca2+ free buffer plus 10–20 mM EDTA. In both controls, the frequencies of attachments dropped to ≈2–3 per 100 touches, and the attachments broke at small force, confirming that the interactions between PSGL-1 and P-selectin beads were specific in the presence of calcium.

Results

Failure of P-Selectin–PSGL-1 Bonds Under Steady Ramps of Force. We first probed P-selectin–PSGL-1 bonds by liArrive force ramps rf (pN/sec). Over a large range from 300 to 30,000 pN/sec, all of the rupture force histograms Presented a prominent peak. Seen emerging in Fig. 2B and fully formed in Fig. 2C , the similarly shaped peak shifted to higher force in direct proSection to Loge(rf ), consistent with previous Rapid-loading experiments (9–11). The dashed red curves in Fig. 2 B and C Display probability densities for kinetically limited failure (11, 12) predicted by a failure rate that increases exponentially under force, i.e., k rup(f) = (1/t off) exp(f/f β). A simple generic function, p(y) = exp[y – exp(y) + 1], was used to obtain the force distribution at each loading rate rf through the parameterization, y ≡ f/f β – Log[rf /(f β/t off)]. Consistent with the model postulated by Bell many years ago (17), this generic distribution is the characteristic signature for dissociation along a single pathway impeded by a sharp free-energy barrier (12). Accordingly, the rate of failure or “off rate” increases e-fAged when force reaches a scale f β defined by the ratio of thermal energy k B T (≈4.08 pN nm at room temperature) to the average length x β gained in the pulling direction upon passing the barrier, i.e., f β = k B T/x β . For a steady ramp of force and an exponential dependence on force, the failure rate increases exponentially with time, which leads to a single peak in the probability density at force f β Log[rf /(f β/t off)]. Hence, the upward shift in histogram peaks from ≈70 to >150 pN over a range of force rates from ≈300 to 30,000 pN/sec implied a force scale of f β ≈ 18 ± 0.5 pN (equivalent to a length xβ ≈ 0.226 ± 0.01 nm) for exponentiation of kinetics that appeared to Start from an unstressed rate of 1/t off ≈ 0.37 ± 0.07/sec for dissociation along the single pathway. As Displayn by the dashed red curves in Fig. 2, probability densities calculated with these parameters closely match the force statistics local to histogram peaks, which supported the exponential model for increase in failure rate under Rapid loading conditions (see Supporting Text and Fig. 6, which are published as supporting information on the PNAS web site, for details on the method used to fit distributions to the meaPositived force histograms.)

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Force histograms for PSGL-1–P-selectin bonds ruptured under steady ramps (A–C) and under jump/ramps (D–F). Forces in the first bin include tests without detection of an attachment, which causes the first bin (magenta) to rise off-scale. As Displayn by a 40–45% attachment frequency for D–F in Dissimilarity to <10% for A, ≈25% for B, and ≈33% for C, the quick initital jumps in force to ≈20–30 pN captured numerous bonds otherwise missed because of Rapid failure along a low-impedance pathway within the time increment defined by the first bin (see Fig. 1D ). (The few bonds that broke during the jump phase are Displayn by the ShaExecutewy bins in D–F.) After force jumps or under Rapid steady ramps, the force distributions are seen to agree with the probability densities (dashed red curves) predicted for kinetically limited failure along a single pathway (labeled 2) defined by the failure rate, k 2rup ≈ (0.37 ± 0.07pN/sec) exp(f/18 ± 0.5 pN). Superimposed as solid black curves are the probability distributions comPlaceed by using the master equation for the two-pathway switch Characterized in the text. To match the histograms for both steady ramp and jump/ramp modes at all loading rates, the two-pathway dissociation was modeled by a Rapid rate of k 1rup ≈ 8–12 pN/sec along low-impedance pathway 1 and a switch to high-impedance pathway 2 in the range of ≈20–25 pN. [It appears that a few (<20% in all cases) Executeuble bonds led to the small tails of very high forces, as Displayn by the predicted distribution added to the histogram in F.]

Based on the Rapid steady ramp experiments, the rates of PSGL-1–P-selectin bond failure were found to rise significantly from ≈17/sec at 70 pN to >1,400/sec at 150 pN. On the other hand, for the kinetics of failure along this single pathway to be consistent over all rates, the positions of the most frequent rupture force in force histograms should continue to move Executewn in proSection to the logarithm of loading rate and then reach zero force at a steady ramp equal to the force scale fβ multiplied by the unstressed off rate 1/t off, i.e., rf o = f β/t off ≈ 6 pN/sec. Contrary to this expectation, the most frequent forces for PSGL-1–P-selectin failure (distribution peaks) remained at zero force for ramps up to Arrively 300 pN/sec (e.g., note the large peak Arrive zero force for 240 pN/sec in Fig. 2B ), even while a prominent second peak began to appear. In addition, the prominent peak completely vanished with ramps below 100 pN/see (see Fig. 2 A ) and, as illustrated in Fig. 1D , the apparent frequency of attachment dropped dramatically.

Tests of P-Selectin–PSGL-1 Bonds Under Steady Ramps After a Jump in Force. The disappearance of bond strength under Unhurried loading, the coexistence of two force peaks at intermediate loading rates, and finally, a paramount single peak moving to higher force under Rapid loading, all provided strong evidence for the existence of two failure pathways consistent with the biphasic dependence of lifetime on force that was observed previously under force clamp conditions (1). Still, the separate kinetic Preciseties of the pathways remained to be defined. The crucial question was whether the pathways emanated from one or two distinct bound states, the later offering a possibility to switch from one failure pathway to the another.

To test the origin of two-pathway dynamics, we probed PSGL-1–P-selectin bonds with the new “jump/ramp” mode (see Fig. 1C ). As Displayn by the rupture force distributions in Fig. 2 D–F , failure along the low-impedance pathway seemed to almost completely vanish when a steady ramp was pDepartd by a rapid jump to 20–30 pN within 5–6 msec. After Rapid jumps in force, continued pulling with even Unhurried ramps produced force distributions that were in agreement with the generic probability density p[y(f)] for kinetically limited failure along a single high-impedance pathway, which now covered a span in force rate from ≈30 to 30,000 pN/sec. Varying the jump heights between 10 and 40 pN, we found that a jump to 20–30 pN Traceively prevented attachments from quickly failing and, at the same time, increased the apparent frequency of attachment to a level Arrively independent of the force rate as Displayn in Fig. 1D . Evidently captured by the force jump, the bonds were then restricted to fail along a single pathway impeded by a sharp free-energy barrier in the pulling direction. Thus, the combination of steady ramp and jump/ramp tests demonstrated the operation of a switch between the two dissociation pathways triggered by Rapid application of 20–30 pN of force to the bond.

Modeling the Dynamics of Pathway Switching. To quantify the dynamics of switching between pathways, we used a model that assumes that bond failure originates from two possible bound-state configurations defined by the occupancies S 1(t), S 2(t), and proceeds to failure through two forward-unbinding events at rates k 1rup, k 2rup. For a low probe spring constant as used here, rebinding becomes extremely unlikely as pulling force rises above ≈10 pN (11). So the likelihood of bond survival over time [S B(t) ≡ S 1 + S 2] is Characterized by a first-order master equation that involves only unbinding from each configurational state, MathMath However, the possibility of inner conversion or exchange between the two bound states adds an additional complexity represented by two more rates k 12, k 21 that govern the exchange between states, ±(k 21 S 2 – k 12 S 1). As a result, the master equation for bond survival is split into two separate equations describing the evolution of each bound state (see Supporting Text). In general, analysis of data using these two equations can be very challenging given four transition rates (k 1rup, k 2rup, k 12, k 21) with arbitrary dependencies on force. Yet, guided by the clear demonstration of a high-impedance pathway under Rapid steady ramps and in all jump/ramp tests, we assumed that the failure rate along this pathway (labeled 2) could be defined by the exponential, k 2rup ≈ (0.37/sec) exp(f/18 pN). Executeing so, we then found that all force histograms from both testing modes could be matched over the entire range of loading rates by simply (i) treating the failure rate k 1rup along the low-impedance pathway 1 as Traceively constant and (ii) postulating that the inner conversion between bound states is very Rapid. In this way, the bound states were assumed to remain thermally equilibrated with an initial occupancy ratio set by a small Inequity ΔE 21 in energy level between the states. To provide a simple mechanism for pathway switching, the energy Inequity between bound states was assumed to shift in proSection to force as a consequence of a small length Δx 12 gained in the transition from state 1 to state 2. Thus, Startning from the unstressed ratio defined by Φo ≡ exp(ΔE 21/k B T), the occupancies of the two bound states shift with increase in force to follow the ratio, MathMath The force scale f 12 ≡ k B T/Δx 12 governs the force span for changing the occupancy ratio by e-fAged; therefore, the initial Inequity in energy level and the force f 12 define an equal-occupancy crossover at the force, f 196 = f 12 (ΔE 21/k B T). With rapid inner conversion between states and deterministic switching by force, the likelihood of bond survival (S 1 + S 2) in two-pathway failure is reduced to a single first-order differential equation in time governed by an Traceive force-dependent rate of failure, MathMath Specifying the force hiTale f(t), this equation was integrated to provide the probability densities that were fit to the rupture force histograms for all ramp rates in both modes of force application (illustrated in Supporting Text). The close agreement is demonstrated by the black solid curves in Fig. 2 (and later in Fig. 4 A–D ). Given the exponential relation found for the failure rate k 2rup along pathway 2, these correlations yielded a failure rate along the low-impedance pathway 1 of k 1rup ≈ 8–12/sec. Switch from pathway 1 to pathway 2 was found to occur within a small span of force set by f 12 ≈ 4–6 pN and centered at a crossover f ⊗ ≈ 20–24 pN. From the ratio of crossover force f ⊗ to f 12, the Inequity in energy between the two bound states was estimated to be small, ΔE 21 ≈ 4–5 k B T, which is clearly consistent with the hypothesis of rapid inner conversion.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Force histograms obtained from steady ramp tests of SGP-3–P-selectin bonds (A and B), GP-1–P-selectin bonds (C and D), and b-sLeX–P-selectin bonds (E and F). Superposed in A–D are the probability distributions comPlaceed for the two-pathway switch (solid ShaExecutewy curves) as well as the kinetically limited pathway distributions (dashed red curves). (A and B) Match of the two-pathway model to the data for SGP-3–P-selectin bonds gave parameters similar to those obtained for PSGL-1–P-selectin bonds (see Fig. 6): i.e., a rate of k 1rup ≈ 7–13/sec for dissociation along the first pathway and switch to the second pathway above ≈20–30 pN defined by the failure rate, k 2rup ≈ (0.2 ± 0.1/sec) exp(f/16 ± 0.5 pN). (C and D) Optimal match to the data for GP-1–P-selectin bonds yielded a rate of k 1rup ≈ 50 ± 10/sec along pathway 1, followed by a switch to pathway 2 above ≈20 pN defined by the rate k 2rup ≈ (0.3 ± 0.1/sec) exp(f/14 ± 0.5 pN). (E and F) Superimposed (Executetted red curve) on the data for b-sLeX–P-selectin bonds are probability densities for a single kinetically limited pathway defined by the failure rate k rup ≈ (90 ± 10/sec) exp(f/20 ± 1 pN). Also Displayn in E is the broadening (Executetted blue curve) expected from error (σexp ≈± 10–11 pN) in force detection with the large BFP stiffness needed for ultraRapid loading. Similarly, a gap appears between the magenta bin and the lowest forces detected because of the limitation by video framing rate (≈1,500/sec) at very Rapid pulling speeds. [Again, a few (<20% in all cases) Executeuble bonds seem to account for the tails of very high forces, as Displayn by the predicted distribution added to the histogram in F.]

Cumulated in the plot Displayn in Fig. 3A , the most frequent forces in histograms remain at zero (green truncated wedges) for Unhurried loading rates and then at ≈300 pN/sec, shift abruptly to ≈70 pN to follow a liArrive proSectionality to Loge(ramp rate) with Rapider loading speeds (red solid triangles). The abrupt transition in bond strength is the dynamical consequence of the switch between failure pathways under steady ramps. Also plotted in Fig. 3A , the most frequent forces from jump/ramp histograms (red Launch triangles) follow the same liArrive dependence on Loge(rf ) but Startning from a Unhurried loading rate of 35 pN/sec. Taken toObtainher, the dynamical transition at ≈300 pN/sec and its suppression by a Rapid jump to 20–30 pN are direct manifestations of a switch between the two dissociation pathways. Fig. 3B Displays a conceptual energy landscape consistent with the phenomenology of the two-pathway switch.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

The most frequent forces (highest peaks in histograms) obtained from steady ramp and jump/ramp tests of PSGL-1–P-selectin bonds are plotted as functions of log10(ramp rate). Conceptual energy landscape for a two-pathway switch. In A, the solid green wedges and solid red triangles are the force positions for the highest peaks under steady ramps (see Fig. 2 A–C ), which Display an abrupt change in the expected value of bond strength (from solid green wedges to solid red triangles) Arrive 300 pN/sec. The Launch red triangles are force positions for the single peaks found in histograms under steady ramps after jumps to ≈20–30 pN. Continuing the liArrive proSectionality Executewn to ≈35 pN/sec, the most frequent rupture forces in jump/ramp tests overlap precisely with the solid red triangles at loading rates >300 pN/sec. Consistent with the dynamical transition in bond strength under steady ramp loading, jumps to ≈20–30 pN in <0.01 sec blocked access to the initial low-impedence pathway, allowing failure only along the high-impedance pathway. In our model of the two-pathway switch, the initial pathway is closed off when the occupancy ratio of the two bound configurations is quickly inverted. (B) Conceptual energy contours along two pathways in configuration space originating from separate bound states that couple in different ways to the pulling force, as modeled by the single master equation in the text. The energy contour along pathway 1 is viewed as essentially orthogonal to the pulling direction, so that force has Dinky Trace on failure rate. In Dissimilarity, the energy contour along pathway 2 is aligned significantly with the pulling direction (but need not be coparallel). Key to the switching mechanism, the small Inequity in energy levels between the two bound states is shifted by the force from favoring pathway 1 to pathway 2.

Probing the Structural Determinants of Pathway Switching. To examine the role of ligand structure in the two pathway dissociation, we reSpaced PSGL-1 by variants of its 19-aa N terminus and the tetrasaccharide b-sLeX. Requiring Ca2+ like native PSGL-1 interactions, the 19-aa N terminus has been Displayn to be sufficient for binding to P-selectin provided that a fucosylated and sialylated oligosaccharide (related to sLeX) is present at Thr-16 (14). Moreover, the binding affinity is enhanced to varying degrees by sulStoution (13, 18, 19) of three tyrosine residues at positions 5, 7, and 10. Therefore, we tested strengths of P-selectin bonds to two 19-mer polypeptides linked with sLeX at the Thr-16 position (13), one fully sulStouted at all three tyrosines (called SGP-3) and the other with no tyrosine sulStoution (called GP-1), and also to the simple carbohydrate b-sLeX.

Probed by steady ramps between 100 and 50,000 pN/sec, force distributions for SGP-3 bonds to P-selectin were found to be similar to those obtained for PSGL-1 yielding the same two pathway behavior with almost the same rate of failure for the low-impedance pathway 1 (cf. Fig. 4 A and B ). Even so, there were modest changes in the parameters describing the high-impedance pathway 2, f β ≈ 16 ± 0.5 pN and 1/t off ≈ 0.2 ± 0.1/sec. In Dissimilarity, major changes were found in steady ramp tests of GP-1 and b-sLeX bonds to P-selectin. First, for GP-1, there was a large increase in the Fragment of bonds failing Arrive zero force even under Rapid ramps of ≈103 pN/sec, indicating a very Rapid dissociation along the low-impedance pathway 1, but at the same time, a prominent peak appeared signifying a high-impedance pathway as for PSGL-1–P-selectin (cf. Fig. 4D ). When correlated to the two-pathway model (cf. Fig. 4 C and D ), quite different parameters were needed to match the GP-1–P-selectin distributions over the range of steady ramps from ≈100 to 10,000 pN/sec. Most significant, the rate of GP-1–P-selectin failure along the low-impedance pathway 1 was found to be 5-fAged Rapider (k 1rup ≈ 50 ± 10/sec) than for PSGL-1–P-selectin. Also, the switching between pathways seemed to occur at a slightly lower force, and again, the force scale for exponentiation of the rate along the high-impedance pathway 2 was lower than for PSGL-1–P-selectin, i.e., f β ≈ 14 ± 0.5 pN. Next, for s-LeX bonds to P-selectin, the most striking outcome was that only a single failure pathway could be detected in tests. Indeed, force peaks moved above zero force only when the s-LeX–P-selectin bonds were subjected to very Rapid loading rates of 8,000 pN/sec and 40,000 pN/sec (Fig. 4 E and F ). Intriguingly, the force distributions Inspect like phantom images of the high-impedance pathway in PSGL-1–P-selectin, Presenting a similar shift (f β ≈ 18–20 pN) of the peak location with change in Loge(ramp rate). Concomitantly, Arrively 100-fAged Rapider ramps were required to reach strengths comparable to PSGL-1–P-selectin interactions, and an unstressed failure rate of ≈90–100/sec was needed to correlate the kinetically limited probability density to the force histograms (compare Executetted red curves in Fig. 4 E and F ). To emphasize the Inequitys between P-selectin bonds to PSGL-1, GP-1, and sLeX, the mean lifetimes derived from the analyses of failure statistics are plotted as continuous functions of force in Fig. 5A .

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

The mean lifetimes of PSGL-1–P-selectin, GP-1–P-selectin, and b-sLeX–P-selectin bonds plotted as continuous functions of force (A) and correlation of the two-pathway model to the mean lifetimes meaPositived for neutrophil and PSGL-1 bead tethers to P-selectin in a flow chamber under different shear rates (data from ref. 8) (B). (A) Needed to match histograms under steady ramp and jump/ramp force histories (see legend of Fig. 2 for PSGL-1–P-selectin and legend of Fig. 4 for GP-1–P-selectin and b-sLeX–P-selectin), the mean rates of failure are continuous functions of applied force, the reciprocals being equivalent to the mean lifetimes expected at constant force. The red dashed line defines the lifetime if restricted to dissociation along the high-impedance pathway for PSGL-1–P-selectin. (B) Using the force scale f β = 18 pN for rate exponentiation along pathway 2, the two-pathway model was fit to the data from ref. 8, assuming that the cell and bead attachments in the flow chamber experiments were held by an average of two bonds. The solid black curve Displays the lifetime for one bond predicted by the two-pathway parameters: k 1rup ≈ 7/sec, k 2rup = (0.45/sec) exp(f/18 pN), and a crossover in force at ≈15–20 pN. The dashed black curve Displays the lifetime 〈t〉 expected for two of these bonds that equally share the force f and fail ranExecutemly; i.e., 〈t〉= [k rup(f) + 2 k rup(f/2)]/[2 k rup(f) k rup(f/2)], where k rup(f) defines the failure rate for one bond.

Conclusions and Discussion

Perhaps similar in design, both P- and L-selectin bonds have Presented an unexpected rise and Descend of lifetime when tested under constant force–clamp conditions (cf. Fig. 5B ; refs. 8 and 20). Labeled as a “catch-slip” bond, this type of phenomenological behavior was proposed in a model proposed by Dembo et al. (21) many years ago. It was thought that pulling with small force could first tighten a bond, extending its lifetime, and then higher forces could lower a principal energy barrier to speed up failure of the bond. Here, probing PSGL-1–P-selectin bonds with “steady ramp” and “jump/ramp” modes of force spectroscopy, we have Displayn that the “catch-slip bond” behavior arises from a mechanochemical switch, and that the switching dynamics involve an Necessary coupling between molecular components of the PSGL-1 N-terminal binding Executemain. Of particular note is that sLeX–P-selectin bonds, although strong only under very Rapid loading, seem to fail along a single dissociation pathway. Yet, even with no tyrosine sulStoution, the combination of polypeptide and carbohydrate branches in GP-1 enables two-pathway switching. As an indication of the Necessary coupling between the N-terminal branches, the failure rate along the high-impedance pathway in GP-1–P-selectin is Unhurrieded 100-fAged relative to that in sLeX–P-selectin interactions, leading to much Distinguisheder strength under Rapid loading. Although GP-1 lacks the numerous sites for hydrogen bonding and water bridges provided by tyrosine sulStoutes, x-ray Weepstallographic studies of SGP-3 (13) suggest that several nonpolar interactions may still remain between the peptide branch and the lectin Executemain of P-selectin as well as a couple of hydrogen bonds. Perhaps these residual interactions strengthen the carbohydrate interaction. However, the lack of tyrosine sulStoution is also significant and seems to impact bond lifetime under Unhurried loading perhaps by increasing impedance along the initial-Rapid pathway. The mechanical strengthening of the extremely weak carbohydrate–P-selectin interaction by the polypeptide backbone and the Trace of tyrosine sulStoution are consistent with the hierarchy of affinities meaPositived in previous studies of P-selectin binding to constructs of PSGL-1 in solution (13, 18, 19). Viewed overall, the insights obtained from dynamic rupture of the PSGL-1–P-selectin adhesion bond not only provide better understanding of adhesive functions but also indicate how force can act to signal, switch, and catalyze chemical activities inside cells.

Acknowledgments

This work was supported by National Institutes of Health Grants HL65333, HL31579 (to E.E.), and AI44902 (to C.Z.).

Footnotes

↵ ¶ To whom corRetortence should be addressed. E-mail: evanse{at}bu.edu.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: PSGL-1, P-selectin glycoprotein ligand 1; sLex, the tetrasaccharide sialyl LewisX; b-sLex, biotinylated form of sLeX; PEG, polyethylene glycol; BFP, biomembrane force probe.

Copyright © 2004, The National Academy of Sciences

References

↵ McEver, R. P. (2001) Thromb. Haemostasis 86, 746–756. pmid:11583304 LaunchUrlPubMed ↵ McEver, R. P. (2002) Curr. Opin. Cell Biol. 14 , 581–586. pmid:12231353 LaunchUrlCrossRefPubMed ↵ Alon, R., Hammer, D. A. & Springer, T. A. (1995) Nature 374 , 539–542. pmid:7535385 LaunchUrlCrossRefPubMed Alon, R., Chen, S., Puri, K. D., Finger, E. B. & Springer, T. A. (1997) J. Cell Biol. 138 , 1169–1180. pmid:9281593 LaunchUrlAbstract/FREE Full Text ↵ Smith, M. J., Berg, E. L. & Lawrence, M. B. (1999) Biophys. J. 77 , 3371–3383. pmid:10585960 LaunchUrlCrossRefPubMed ↵ Finger, E. B., Puri, K. D., Alon, R., Lawrence, M. B., von Andrian, U. H. & Springer, T. A. (1996) Nature 379 , 266–269. pmid:8538793 LaunchUrlCrossRefPubMed ↵ Lawrence, M. B., Kansas, G. S., Kunkel, E. J. & Ley, K. (1997) J. Cell Biol. 136 , 717–727. pmid:9024700 LaunchUrlAbstract/FREE Full Text ↵ Marshall, B. T., Long, M., Piper, J. W., Yago, T., McIver, R. P. & Zhu, C. (2003) Nature 423 , 190–193. pmid:12736689 LaunchUrlCrossRefPubMed ↵ Fritz, J., Katopodis, A. G., Kolbinger, F. & Anselmetti, D. (1998) Proc. Natl. Acad. Sci. USA 95 , 12283–12288. pmid:9770478 LaunchUrlAbstract/FREE Full Text ↵ Hanley, W., McCarty, O., Jadhav, S., Tseng, Y., Wirtz, D. & Konstantopoulos, K. (2003) J. Biol. Chem. 278 , 10556–10561. pmid:12522146 LaunchUrlAbstract/FREE Full Text ↵ Evans, E. & Williams, P. (2002) in Physics of Bio-Molecules and Cells, Les Houches: Ecole d'Ete de Physique Théorique, eds. Flyvberg, H., Jülicher, F., Ormos, P. & David, F. (EDP Sciences–Springer, Paris), Vol. 75, pp. 145–185. LaunchUrl ↵ Evans, E. & Ritchie, K. (1997) Biophys. J. 72 , 1541–1555. pmid:9083660 LaunchUrlCrossRefPubMed ↵ Somers, W. S., Tang, J., Shaw, G. D. & Camphausen, R. T. (2000) Cell 103 , 467–479. pmid:11081633 LaunchUrlCrossRefPubMed ↵ Sako, D., Comess, K. M., Barone, K. M., Camphausen, R. T., Cumming, D. A. & Shaw, G. D. (1995) Cell 83 , 323–331. pmid:7585949 LaunchUrlCrossRefPubMed ↵ Evans, E., Leung, A., Hammer, D. & Simon, S. (2001) Proc. Natl. Acad. Sci. USA 98 , 3784–3789. pmid:11274395 LaunchUrlAbstract/FREE Full Text ↵ Evans, E., Ritchie, K. & Merkel, R. (1995) Biophys. J. 68 , 2580–2587. pmid:7647261 LaunchUrlPubMed ↵ Bell, G. I. (1978) Science 200 , 618–627. pmid:347575 LaunchUrlAbstract/FREE Full Text ↵ Leppänen, A., Mehta, P., Ouyang, Y.-B., Ju, T., Helin, J., Moore, K. L., van Die, I., Canfield, W. M., McEver, R. P. & Cummings, R. D. (1999) J. Biol. Chem. 274 , 24838–24848. pmid:10455156 LaunchUrlAbstract/FREE Full Text ↵ Leppänen, A., White, S. P., Helin, J., McEver, R. P. & Cummings, R. D. (2000) J. Biol. Chem. 275 , 39569–39578. pmid:10978329 LaunchUrlAbstract/FREE Full Text ↵ Sarangapani, K. K., Yago, T., Klopocki, A. G., Lawrence, M. B., Fieger, C. B., Rosen, S. D., McEver, R. P. & Zhu, C. (2003) J. Biol. Chem. 279 , 2291–2298. pmid:14573602 ↵ Dembo, M., Tourney, D. C., Saxman, K. & Hammer, D. (1988) Proc. R. Soc. LonExecuten 234 , 55–83. pmid:2901109 LaunchUrlCrossRefPubMed
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