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 high-affinity, 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. Significance Rolling is a first step for cells to transmigrate across the endothelium and is mediated by specialized adhesion receptors. Although it has been demonstrated that the mechanical force positively regulates rolling mediated by selectins and integrins, it remains elusive how force affects the rolling mediated by a hyaluronan receptor, CD44. Here, we demonstrate that the force applied from the C terminus of the hyaluronan-binding domain of CD44 stabilized the CD44-mediated rolling. We also found that the effect of force is to shorten the transition time from the low- to the high-affinity state. The mechanism described here provides the structural basis for the CD44-mediated rolling, which is important for lymphocyte trafficking and the stem cell homing.