An Isoleucine-based Allosteric Switch Controls Affinity and Shape Shifting in Integrin CD11b A-domain

Abstract

In response to cell activation signals, integrins switch from a low to a high affinity state. Physiologic ligands bind to integrins through a von Willebrand Factor Atype domain. Crystallographic studies revealed two conformations of this domain, "closed" and "open." The latter crystallizes in complex with a pseudoligand or ligand, suggesting that it represents the high affinity state; data linking structure and activity are lacking however. In this communication, we expressed stable low and high affinity forms of integrin CD11b A-domain and determined their binding isotherms and crystal structures. The low affinity form, generated by deleting an N-terminal extension extrinsic to the domain, did not bind to physiologic ligands, and crystallized in the closed conformation. The high affinity form was generated by either deleting or substituting an invariable C-terminal Ile 316 , wedged into a hydrophobic socket in the closed form, but displaced from it in the open structure. Both mutants crystallized in the open conformation, and the Ile 316 3 Gly-modified integrin displayed high affinity. Structural differences between the low and high affinity forms were detected in solution. These data establish the structure-function correlates for the CD11b A-domain, and define a ligand-independent isoleucine-based allosteric switch intrinsic to this domain that controls its conformation and affinity.Integrins are heterodimeric receptors that mediate vital cellcell and cell-matrix adhesive interactions (1). Integrins bind to physiologic ligands in a divalent cation-dependent manner and require a solvent-exposed acidic residue in their respective ligands for binding. Integrin interactions with physiologic ligands are dependent on inside-out signals, which switch integrins from a "low" to a high affinity state (2). This functional up-regulation is associated with conformational changes in the extracellular regions of integrins (2) that involve two major ligand binding sites, a von Willebrand Factor (vWF) 1 A-type domain (named A-or I-domain) present in the ␣ subunits of nine integrins, and an A-like domain embedded in all eight integrin ␤ subunits (3, 4).The integrin A-domain assumes a dinucleotide-binding fold (3,(5)(6)(7)(8), with a metal ion-dependent adhesion site (MIDAS) on one end (defined as the top of the domain) and is connected through the adjacent N and C termini on the opposite end (bottom) to the body of the integrin. MIDAS and its surrounding surface-exposed side chains form the binding site for several physiologic ligands (4, 6, 9 -12) and for the antagonist, neutrophil inhibitory factor, NIF (13,14). The A-domain has been crystallized in two conformations, "open" and "closed". In the open conformation, three noncharged residues in the protein directly coordinate the metal ion in MIDAS; a pseudoligand or ligand glutamate residue (3, 6, 15) completes metal coordination. In the closed form, the amphipathic C-terminal ␣7 helix is shifted upwards by 10 Å. This large shift is associated with a change in metal coordinat...

Exposure of acidic residues as a danger signal for recognition of fibrinogen and other macromolecules by integrin αXβ2

“…The differences between ␣ M ␤ 2 and ␣ X ␤ 2 were mirrored in SPR assays with ␣ M and ␣ X I domains stabilized in their active conformations (20,26), where the ␣ X I domain generated a severalfold higher response than ␣ M I domain in the binding to Fg (Fig. 3 A and B).…”

Modulation of CD11b/CD18 Adhesive Activity by Its Extracellular, Membrane-Proximal Regions

“…However, the molecular mechanism underlying integrin activation remains ill defined. Studies over the past several years have been focused mainly on the cytoplasmic domain and the ligand binding domain of the integrin receptor (16,17,24,25), whereas the region that bridges these two functional domains has not been well studied. In this work we investigated the role of this transitional region, specifically the extracellular, membrane-proximal regions of CD11b/CD18, on receptor activation, receptor clustering, and cell surface distribution.…”

“…Previous studies of "inside-out" signaling have focused mainly on the cytoplasmic domain (16,17) and the ligand binding domain of integrins (24,25). However, the role of the transitional region between these two domains, especially the extracellular, membraneproximal region of the integrin receptor, in controlling integrin functions has not been probed.…”

Integrin αDβ2, an adhesion receptor up-regulated on macrophage foam cells, exhibits multiligand-binding properties

“…19,35,37 The high-affinity form of the ␣ M I-domain can be generated by unlocking the C-terminal Ile 316 from a hydrophobic socket either by mutation or truncation the ␣7 C-terminal helix at Lys 315 . 35 The fact that the ␣ D I-domain contains an invariable Ile in the 311 LKEKIFA 317 sequence, which is highly homologous to the ␣ M 312 LREKIFA 318 , and the observation that truncation of the ␣ D I-domain at Lys 314 converts it into the active form, suggest that the ␣ D I-domain undergoes conformational changes similar to those of the ␣ M I-domain. The I-domains of the ␣ M , ␣ L , and ␣ X subunits have been crystallized in both "open" and "closed" conformations that correspond to the high-and lowaffinity states, respectively.…”