The connection between metal ion affinity and ligand affinity in integrin I domains

Abstract

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.

Multiple low-affinity interactions support binding of human osteopontin to integrin α X β 2

“…In SPR experiments these ligands bind the α X I domain with remarkably similar binding kinetics, but differ considerably in apparent binding stoichiometry. This is a type of ligand recognition unlike what has been reported for the highly similar α L β 2 and α M β 2 integrins and their ligands [18,22,49,50]. Nor is this type of recognition similar to what was reported for other integrins, which bind specific, non-repeated motifs in OPN.…”

“…While this influence of Mn 2+ involves the beta-chain I-like domain [53], we cannot exclude, however, the possibility that Mn 2+ also bind in the α X I domain MIDAS. Evidence from the wild-type α L and α M I domains suggests that Mn 2+ ions bind~5-15 fold stronger than the physiologically relevant Mg 2+ ions [50,54], but it is not clear if this alters in any way the ligand interaction by the I domain. As demonstrated by the lack of binding to HCM in these experiments compared to the robust binding to OPN, however, the Mn 2+ -activated α X β 2 integrins are capable of discriminating between ligands and non-ligands.…”

LFA-1 Affinity Regulation Is Necessary for the Activation and Proliferation of Naive T Cells

“…2D7 binds to the N terminus of the I-domain (residues 118 -153), which encompasses most of the metal ion-dependent adhesion site-coordinating residues. Changes in the metal ion-dependent adhesion site that accompany activation may result in an effective conformation-sensitive epitope for LFA-1 (48). Thus, the epitope of 2D7 is conformation-sensitive, and the binding is abolished when LFA-1 is locked in the high affinity conformation.…”

Shedding of Large Functionally Active CD11/CD18 Integrin Complexes from Leukocyte Membranes during Synovial Inflammation Distinguishes Three Types of Arthritis through Differential Epitope Exposure

“…The monovalent interaction between CD11a/CD18 and ICAM-1 has been investigated in many studies through application of the ligand binding I domain of CD11a/CD18 mutated to assume the high-affinity conformation. Several strategies for stabilizing this high-affinity conformation reported K D values for the interaction of 200 nM (62,63) in a mode of binding consistent with a 1:1 stoichiometry (64), as expected from structural studies of the binding (63). However, the high affinity of the sCD11/CD18 complexes for ICAM-1 in our assays exceed the affinity of the monovalent interaction with as much as 10 8 -fold using the halfmaximum saturating concentration of sCD11/CD18 as an approximation of the K D for the interaction.…”

“…Similarly, the presence of more than one ligand binding domain in the complexes likely supports avidity binding to immobilized ICAM-1. With the monovalent affinity (62,63) of the CD11a chain I domain for ICAM-1 the dissociation constant for avidity binding for a 23(CD11a/ CD18) complex would be in the order of 10 214 M, clearly on a scale that would support significant fractional coverage of ICAM-1 ligand binding sites with the plasma dilutions used. A recent report indicated that activated MMP-9 is important in the shedding of CD11a/CD18 from a murine macrophage cell line (7).…”