Remodeling of the lectin–EGF-like domain interface in P- and L-selectin increases adhesiveness and shear resistance under hydrodynamic force

Abstract

Crystal structures of the lectin and epidermal growth factor (EGF)-like domains of P-selectin show 'bent' and 'extended' conformations. An extended conformation would be 'favored' by forces exerted on a selectin bound at one end to a ligand and at the other end to a cell experiencing hydrodynamic drag forces. To determine whether the extended conformation has higher affinity for ligand, we introduced an N-glycosylation site to 'wedge open' the interface between the lectin and EGF-like domains of P-selectin. This alteration increased the affinity of P-selectin for its ligand Pselectin glycoprotein 1 (PSGL-1) and thereby the strength of P-selectin-mediated rolling adhesion. Similarly, an asparagine-to-glycine substitution in the lectin-EGF-like domain interface of L-selectin enhanced rolling adhesion under shear flow. Our results demonstrate that force, by 'favoring' an extended selectin conformation, can strengthen selectin-ligand bonds.Leukocyte migration to inflamed tissues and lymphocyte homing to peripheral lymphoid organs involves a multistep process 1-6 . In the first step, leukocytes tether and roll along vascular endothelia. Rolling exposes leukocytes to chemokines that activate integrins, which arrest leukocyte rolling and mediate leukocyte migration across the endothelia.Rolling results from the hydrodynamic drag force acting on adherent cells. For rolling to be stable, the formation of new receptor-ligand bonds downstream must balance the dissociation of bonds upstream. Leukocyte tethering and rolling are mediated mainly by E-, L-and Pselectins. By initiating transient, rapidly reversible receptor-ligand interactions, these C-type lectin molecules allow a 'zone of adhesion' to move along a vessel wall 7-9 . In contrast, antibody-antigen interactions are unable to support stable rolling over a wide range of shear stresses 10 .Two features contribute to the ability of selectins to mediate stable leukocyte rolling. First, force increases the number of bonds that form between a rolling cell and its substrate, which effectively compensates for the shortening of receptor-ligand bond lifetimes that occurs as force on the bond increases 11 . Cell deformation and tether extension also promote leukocyte rolling 11-13 . Second, individual selectin-ligand bonds are more resistant to force than any other measured receptor-ligand bond 8,11,14 . As force on the receptor-ligand bond increases, the 'off rate' (k off ) increases less for selectin-ligand bonds than for integrin-ligand orCorrespondence should be addressed to T.A.S. (springeroffice@cbr.med.harvard.edu).. Note: Supplementary information is available on the Nature Immunology website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. Selectins contain an N-terminal calcium-dependent lectin domain, an epidermal growth factor (EGF)-like domain, a variable number of short consensus repeats (SCRs), and transmembrane and cytoplasmic domains 6,18-20 . Selectin ligands consist mainly of mucin-like sialoglycopro...

Transmission of allostery through the lectin domain in selectin-mediated cell adhesion

“…A mutation introduced into the lectin-EGF interface designed to favor the extended, ligand-bound conformation of P-selectin does indeed exhibit a higher affinity for ligand in solution, and when expressed on the cell surface results in slower cell rolling velocities and increased resistance to shear stress (13). This finding provides support for the role of a conformational change in selectin function.…”

“…This finding provides support for the role of a conformational change in selectin function. Mutations at the lectin-EGF interface in L-selectin also altered its rolling behavior (13).…”

“…It should be pointed out that the bent conformation crystallized in the absence of ligand can also bind ligand, as shown by soaking ligand into preformed crystals (11). The conformation crystallized in the presence of ligand is more extended, because the distance between the ligand binding site and the C terminus of the EGF domain, where tensile force would be applied in vivo, is greater than in the bent conformation.Extended protein conformations are favored by applied tensile force, and if the extended conformation is higher affinity, this provides a mechanism for explaining catch-bond behavior (12)(13)(14). A mutation introduced into the lectin-EGF interface designed to favor the extended, ligand-bound conformation of P-selectin does indeed exhibit a higher affinity for ligand in solution, and when expressed on the cell surface results in slower cell rolling velocities and increased resistance to shear stress (13).…”

“…Extended protein conformations are favored by applied tensile force, and if the extended conformation is higher affinity, this provides a mechanism for explaining catch-bond behavior (12)(13)(14). A mutation introduced into the lectin-EGF interface designed to favor the extended, ligand-bound conformation of P-selectin does indeed exhibit a higher affinity for ligand in solution, and when expressed on the cell surface results in slower cell rolling velocities and increased resistance to shear stress (13).…”

Selectin catch–slip kinetics encode shear threshold adhesive behavior of rolling leukocytes

“…Unlike L-selectin, a shear threshold is not consistently observed for leukocytes adhering to P-or E-selectin (2-4), even though both share homologous structures with L-selectin, and P-selectin has been extensively characterized as a bona fide catch bond (13,16). Recent attempts to understand these differences by using site-directed mutagenesis and selectin chimeras have revealed a role for allosteric regulation at the interdomain hinge linking the lectin headpiece to the EGFlike stalk (17,18). Greater hinge flexibility appears to be associated with a transition to the resilient molecular conformation at lower levels of shear stress and leads to an overall enhancement in rolling adhesion.…”