The Effects of Load on E-Selectin Bond Rupture and Bond Formation

Abstract

Molecular dissociation rates have long been known to be sensitive to applied force. We use a laser trap to provide evidence that rates of association may also be force-dependent. We use the thermal fluctuation assay to study single bonds between E-selectin and sialyl Lewis(a) (sLe(a)), the sugar on PSGL-1 to which the three selectins bind. Briefly, an E-selectin-coated bead is held in a laser trap and pressed with various compressive loads against the vertical surface of a bead coated with sLe(a). The time it takes for a bond to form is used to calculate a specific two-dimensional on-rate, kono. We observe an increase in kono with increasing compressive force, providing single molecule evidence that on-rate, in addition to off-rate, is influenced by load. By measuring bond lifetimes at known tensile loads, we show that E-selectin, like its family members L- and P-selectin, is capable of forming catch bonds. Our data support a reverse Bell model, in which compressive forces lower the activation energy for binding. Load-dependent on-rates may be a general feature of all intermolecular bonds.

FIGURE 1

Experimental setup and representative laser trap data of a bond event. Letters in the data trace correspond with similarly lettered panels in the experimental setup diagrams. The trapped bead is stepped toward the stationary bead (a), a bond forms (b), the trapped bead is stepped away from the stationary bead (c), a bond ruptures (d), and if there was a second bond present, a second bond ruptures (e). Measurements of t_on and t_off are used to determine on-rate and off-rates, respectively. In the case of multiple bonds, the lifetime of the n_th bond is designated t_offn.

FIGURE 2

(a) The fraction of bead contacts forming bonds (●) as a function of sLe^a concentration (top x-axis) and sLe^a site density (bottom x-axis). (b) The fraction of bonds that are single bonds (●) and multiple bonds (○) as a function of sLe^a concentration and sLe^a site density.

FIGURE 3

Bond lifetime as a function of applied load for single bonds. Data is fit by the two-pathway model for catch–slip adhesion. Error bars indicate standard error of the mean.

FIGURE 4

Time to bond formation (t_on) as a function of compressive force. Data fit by two-state conformational change model (blue line), tilting energy landscape model (red), and surface layer deformation model (green). Error bars indicate standard error of the mean.

FIGURE 5

(a) A schematic for the load-dependent association model integrated into the two-state model. Compressive force shifts the equilibrium of unbound receptors to conformational state R₂, which has a faster association rate than state R₁. Tensile force shifts the equilibrium of bound complex to state RL₂, which has a slower dissociation rate than state RL₁. In the compressive regime (top half of figure), +f indicates compressive force, while in the tensile regime, +f indicates tensile force. (b) A schematic for the reverse Bell model. Compressive force increases the rate at which bonds form, while tensile force decreases the rate at which bonds rupture.