The observed stability of a protein is altered when ligands bind, which results in a shift in the melting temperature (T(m)). Binding to the native state in the absence of binding to the denatured state will necessarily lead to an increase in the T(m), while binding to the unfolded state in the absence of native state binding will decrease the T(m) relative to that of the protein in the absence of ligand. These effects are required by the thermodynamics of reversible folding. However, the relationship between binding affinity and the magnitude of the observed temperature shift is not a simple correlation (i.e., a larger shift in T(m) does not necessarily mean tighter binding) and is complicated by interaction with the denatured state. Using exact simulations, the range of behavior for the dependence of the observed T(m) shift on the energetics of ligand binding is investigated here. Specifically, differential scanning calorimetry (DSC) curves are simulated for protein unfolding in the presence of ligands binding to both the native and denatured states. The results have implications for drug screening and the determination of heat capacity changes for protein unfolding.

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...

The selectins are cell adhesion proteins that must resist applied forces to mediate leukocyte tethering and rolling along the endothelium and have 2 conformational states. Selectin-ligand bond dissociation increases only modestly with applied force, and exhibits catch bond behavior in a low-force regime where bond lifetimes counterintuitively increase with increasing force. Both allosteric and sliding-rebinding models have emerged to explain catch bonds. Here, we introduce a large residue into a cleft that opens within the lectin domain to stabilize the more extended, high-affinity selectin conformation. This mutation stabilizes the high-affinity state, but surprisingly makes rolling less stable. The position of the mutation in the lectin domain provides evidence for an allosteric pathway through the lectin domain, connecting changes at the lectin-EGF interface to the distal binding interface.cell rolling ͉ mechanochemistry ͉ vasculature I n an inflammatory response, leukocytes in the bloodstream first tether and roll along the endothelium, then arrest and extravasate through the vessel wall to the site of infection or injury (1, 2). The ability of leukocytes to tether and roll over the wide range of shear stresses experienced in the vasculature is mediated by cell surface-displayed lectins referred to as the selectins. P-selectin is expressed on activated endothelium and platelets, and its primary ligand P-selectin glycoprotein ligand 1 (PSGL-1), which is expressed on leukocytes (2). The processes of cell tethering, rolling, and arrest have all been reproduced by using in vitro flow chambers.Rolling through selectins is unusually stable to changes in the concentration of ligand on the substrate and the wall shear stress. As wall shear stress increases, rolling velocity increases much less. One factor contributing to the mechanical stability of rolling through selectins is the relatively moderate increase in the off rates for selectin-ligand bonds as the force experienced by the bond is increased (3-5). Unique to the selectins among leukocyte adhesion molecules is the observation of a shear threshold effect for cell tethering and rolling adhesion. At low shear stresses few cells tether and rollingly adhere, whereas above a threshold shear stress (or shear), many cells tether and roll. With increasing shear, more and more cells tether and roll, until a peak is reached, beyond which increasing shear results in decreased numbers of rolling cells. This effect was first demonstrated for L-selectin (6) and later for P-and E-selectin (7). Measurements of the number of bonds between a rolling cell and the substrate have provided an explanation for these effects (8). More bonds are present being a rolling cell and the substrate at high shear than low shear. The shear threshold occurs at the shear where the number of bonds between the cell and the substrate is close to 1. Thus, as wall shear stress increases, the higher rate of breakage of individual selectin-ligand bonds is largely compensated by the formation of a large...

Integrins are cell-surface heterodimeric proteins that mediate cell-cell, cell-matrix, and cell-pathogen interactions. Half of the known integrin alpha subunits contain inserted domains (I domains) that coordinate ligand through a metal ion. Although the importance of conformational changes within isolated I domains in regulating ligand binding has been reported, the relationship between metal ion binding affinity and ligand binding affinity has not been elucidated. Metal and ligand binding by several I domain mutants that are stabilized in different conformations are investigated using isothermal titration calorimetry and surface plasmon resonance studies. This work suggests an inverse relationship between metal ion affinity and ligand binding affinity (i.e. constructs with a high affinity for ligand exhibit a low affinity for metal). This trend is discussed in the context of structural studies to provide an understanding of interplay between metal ion binding and ligand affinities and conformational changes.