Mechanical force effect on the two-state equilibrium of the hyaluronan-binding domain of CD44 in cell rolling

CD44 is the receptor for hyaluronan (HA) and mediates cell rolling under fluid shear stress. The HA-binding domain (HABD) of CD44 interconverts between a low-affinity, ordered (O) state and a highaffinity, partially disordered (PD) state, by the conformational change of the C-terminal region, which is connected to the plasma membrane. To examine the role of tensile force on CD44-mediated rolling, we used a cell-free rolling system, in which recombinant HABDs were attached to beads through a C-terminal or N-terminal tag. We found that the rolling behavior was stabilized only at high shear stress, when the HABD was attached through the C-terminal tag. In contrast, no difference was observed for the beads coated with HABD mutants that constitutively adopt either the O state or the PD state. Steered molecular dynamics simulations suggested that the force from the C terminus disrupts the interaction between the C-terminal region and the core of the domain, thus providing structural insights into how the mechanical force triggers the allosteric O-to-PD transition. Based on these results, we propose that the force applied from the C terminus enhances the HABD-HA interactions by inducing the conformational change to the high-affinity PD transition more rapidly, thereby enabling CD44 to mediate lymphocyte trafficking and hematopoietic progenitor cell homing under high-shear conditions.