Abstract
Integrins are transmembrane cell-extracellular matrix adhesion receptors that impact many cellular functions. A subgroup of integrins contain an inserted (I) domain within the α–subunits (αI) that mediate ligand recognition where function is contingent on binding a divalent cation at the metal ion dependent adhesion site (MIDAS). Ca2+ is reported to promote α1I but inhibit α2I ligand binding. We co-crystallized individual I-domains with MIDAS-bound Ca2+ and report structures at 1.4 and 2.15 Å resolution, respectively. Both structures are in the “closed” ligand binding conformation where Ca2+ induces minimal global structural changes. Comparisons with Mg2+-bound structures reveal Mg2+ and Ca2+ bind α1I in a manner sufficient to promote ligand binding. In contrast, Ca2+ is displaced in the α2I domain MIDAS by 1.4 Å relative to Mg2+ and unable to directly coordinate all MIDAS residues. We identified an E152-R192 salt bridge hypothesized to limit the flexibility of the α2I MIDAS, thus, reducing Ca2+ binding. A α2I E152A construct resulted in a 10,000-fold increase in Mg2+ and Ca2+ binding affinity while increasing binding to collagen ligands 20%. These data indicate the E152-R192 salt bridge is a key distinction in the molecular mechanism of differential ion binding of these two I domains.
Highlights
Integrins are widely expressed cell surface receptors that couple cell- extracellular matrix (ECM) interactions with the cytoskeleton and transduce mechanochemical signals across the plasma membrane initiating a biological response
Metal binding to the integrin β-subunit is required for allosteric signal transduction where the Mg2+-binding “I-like” domain metal ion dependent adhesion site (MIDAS) is flanked on either side by Ca2+-binding adjacent to MIDAS (ADMIDAS) and synergistic metal-binding sites (SYMBS)[22,23,24,25,26]
Both α1-I domain (α1I) and α2-I domain (α2I) are in the “closed” ligand binding conformation
Summary
Integrins are widely expressed cell surface receptors that couple cell- extracellular matrix (ECM) interactions with the cytoskeleton and transduce mechanochemical signals across the plasma membrane initiating a biological response. An essential feature of the integrin activation mechanism is a force-bearing metal bond linking integrin α-subunit I domains (αI) with physiological ligands (Fig. 1B)[9]. Non-conserved residues surrounding the MIDAS contribute to ligand specificity (Fig. 1C)[10,11,12,13,14,15,16,17,18,19,20,21]. Mutagenesis studies have demonstrated the impact of I domain modifications on overall integrin activation, ligand binding, and subsequent function[40,41,42,43]. Α1I and α2I have a differential response to select metal cofactors whose mechanism of action remains unknown
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