A Minimized Human Integrin α5β1 That Retains Ligand Recognition

Abstract

Two isolated recombinant fragments from human integrin, and induces a specific conformational adaptation of the fibronectin ligand. A two-site model for RGD binding to both ␣ and ␤ integrin components is inferred from our data using low molecular weight RGD mimetics. Integrins (IN)1 are a family of structurally and functionally related adhesion receptors that participate in cell-cell and cellextracellular matrix (EM) interactions (1). All integrins are heterodimers of non-covalently associated ␣ and ␤ subunits. At the functional level, the interactions between integrins and their EM protein ligands involve the following: (i) the extracellular integrin heteromeric "head" that encompasses the Nterminal halves from both ␣ and ␤ subunits and hosts the ligand-recognition pocket with a variety of binding sites on each subunit (2); (ii) short specific amino acid sequences (adhesion motifs) from the EM ligands. The prototype for these adhesion motifs is the Arg-Gly-Asp (RGD) sequence that is present in fibronectin, fibrinogen, vitronectin, and other EMadhesive proteins (3). The exact location of these binding loci in the integrin subunits, as well as their respective role on ligand binding energy and specificity, still remains an open question. The N-terminal half of the integrin ␣ subunits is characterized by the presence of seven N-terminal repeats of about 60 amino acids each (4, 5). Some of the ␣ subunits include an insertion domain, or I-domain, about 200 residues in length, between repeats II and III (2). The homologies between repeats I and VII essentially include the FG and GAP consensus sequences, so that these repeats are also referred as FG-GAP repeats (6). Three to four of these repeats (i.e. repeat IV or V to repeat VII) display sequences that resemble the EF-hand consensus sequence found in various divalent cation-binding proteins (7). However, the integrin EF-hand type sequences are systematically devoid of an acidic residue at their relative position 12, a highly invariant Glu residue in the typical EF-hands, that is replaced by a non-polar residue in integrins (8). Isolated recombinant integrin fragments encompassing repeats III to VII of ␣ IIb (9) and IV to VII of ␣ 5 (10) have been shown to mimic the ligand-binding features (divalent cation and RGD dependence) that are observed with native integrin receptors, indicating that the divalent cation-binding domains in the ␣ subunits are part of the ligand-recognition pocket of integrins. Repeats III and IV in the ␣ subunits have also been shown to be involved in cell spreading and in assembly of focal contacts at the cell surface (11).The N-terminal regions of the integrin ␤ subunits are characterized by a conserved domain that displays sequence homology with the I-domain found in several integrin ␣ subunits (12). This I-type domain in the integrin ␤ subunits includes the following: (i) a functional cation-binding site that displays strong similarities at the level of its metal-coordinating residues with the MIDAS site of the ␣ subunit I-domains (13, 14); ...

The rbmBCDEF Gene Cluster Modulates Development of Rugose Colony Morphology and Biofilm Formation in Vibrio cholerae

“…The InterPro database indicated that both RbmC and Bap1 contain one possible calcium-binding site, two and one FG-GAP domains, respectively, and one galactose mutarotase-like domain. The FG-GAP domains are found in the extracellular N-terminal region of the integrin ␣-chain, which has been reported to be involved in ligand recognition and binding with proteins in the extracellular matrix or surface proteins on other cells (5,43). The galactose mutarotase-like domains, on the other hand, are described by the Structural Classification of Proteins database as probable carbohydrate-binding domains in enzymes acting on sugars.…”

Structure-Function of the Putative I-domain within the Integrin β2 Subunit

“…Recently, a novel cation binding site, termed the MIDAS motif, was identified in the crystal structures within the I-domains of several ␣ subunits and was found to be central to ligand binding functions of these I-domains (10, 58 -61). Evidence for the existence of MIDAS motifs in the I-domains of ␤ 1 , ␤ 2 , and ␤ 3 has also been developed (14,16,17,47,51,62,64). Although the I-domains of the ␣ and ␤ subunits are predicted to have similar MIDAS folds, their cation binding properties appear to differ significantly.…”

“…This region of ␤ 1 (residues 121-329) forms a heterodimer with ␣ 5 (160 -448) (47), and the same region of ␤ 3 (residues 111-318) complexes with ␣ IIb (1-233) (51 V 275 GSDNH between human and avian ␤ 3 was found to change the specificity of ␣ IIb /␤ 3 association (51). Taken together, these data strongly implicate this central region of the ␤ subunits in either heterodimer formation or in controlling the pairing specificity between the ␣ and ␤ subunits.…”

“…Although the I-domains of the ␣ and ␤ subunits are predicted to have similar MIDAS folds, their cation binding properties appear to differ significantly. Using several different approaches including x-ray crystallography, circular dichroism, and fluorescence, it appears that cation binding to the I-domains of the ␣ subunits and the ␤ 1 , ␤ 3 , and ␤ 5 subunits can lead to changes in conformation and ligand binding activity (14,44,45,47,50,63). Compared with the ␤ 1 and ␤ 3 subunits, the role of cation binding in controlling the conformation of the ␤ 2 subunit is not well understood.…”

The Synergy Peptide PHSRN and the Adhesion Peptide RGD Mediate Cell Adhesion through a Common Mechanism

“…Early studies showed that PHSRN binds to the R 5 subunit (20,23) while RGD binds to both the R 5 and 1 subunits (45)(46)(47)(48). It was also demonstrated that PHSRN is not required for cell adhesion to FN and FN matrix assembly when integrins are in the activation state (23,49).…”