Jian-Ping Xiong

Coming to grips with integrin binding to ligands

Integrins are alphabeta heterodimeric cell-surface receptors that are vital to the survival and function of nucleated cells. They recognize aspartic-acid- or a glutamic-acid-based sequence motifs in structurally diverse ligands. Integrin recognition of most ligands is divalent cation dependent and conformationally sensitive. In addition to this common property, there is an underlying binding specificity between integrins and ligands for which there has been no structural basis. The recently reported crystal structures of the extracellular segment of an integrin in its unliganded state and in complex with a prototypical Arg-Gly-Asp (RGD) ligand have provided an atomic basis for cation-mediated binding of aspartic-acid-based ligands to integrins. They also serve as a basis for modelling other integrins in complex with larger physiologic ligands. These models provide new insights into the molecular basis for ligand binding specificity in integrins and its regulation by activation-driven tertiary and quaternary changes.

Three-dimensional EM structure of the ectodomain of integrin αVβ3 in a complex with fibronectin

Integrins are αβ heterodimeric cell surface receptors that mediate transmembrane signaling by binding extracellular and cytoplasmic ligands. The ectodomain of integrin αVβ3 crystallizes in a bent, genuflexed conformation considered to be inactive (unable to bind physiological ligands in solution) unless it is fully extended by activating stimuli. We generated a stable, soluble complex of the Mn2+-bound αVβ3 ectodomain with a fragment of fibronectin (FN) containing type III domains 7 to 10 and the EDB domain (FN7-EDB-10). Transmission electron microscopy and single particle image analysis were used to determine the three-dimensional structure of this complex. Most αVβ3 particles, whether unliganded or FN-bound, displayed compact, triangular shapes. A difference map comparing ligand-free and FN-bound αVβ3 revealed density that could accommodate the RGD-containing FN10 in proximity to the ligand-binding site of β3, with FN9 just adjacent to the synergy site binding region of αV. We conclude that the ectodomain of αVβ3 manifests a bent conformation that is capable of stably binding a physiological ligand in solution.

Crystal structure of the complete integrin αVβ3 ectodomain plus an α/β transmembrane fragment

We determined the crystal structure of 1TM-αVβ3, which represents the complete unconstrained ectodomain plus short C-terminal transmembrane stretches of the αV and β3 subunits. 1TM-αVβ3 is more compact and less active in solution when compared with ΔTM-αVβ3, which lacks the short C-terminal stretches. The structure reveals a bent conformation and defines the α–β interface between IE2 (EGF-like 2) and the thigh domains. Modifying this interface by site-directed mutagenesis leads to robust integrin activation. Fluorescent lifetime imaging microscopy of inactive full-length αVβ3 on live cells yields a donor–membrane acceptor distance, which is consistent with the bent conformation and does not change in the activated integrin. These data are the first direct demonstration of conformational coupling of the integrin leg and head domains, identify the IE2–thigh interface as a critical steric barrier in integrin activation, and suggest that inside-out activation in intact cells may involve conformational changes other than the postulated switch to a genu-linear state.

Does the Integrin αA Domain Act as a Ligand for its βA Domain

We thank Nancy Hogg for providing mAb 24 and Martyn K. Robinson for the KIM185 and 127 mAbs. This research was supported by grants from the National Institutes of Health (NIDDK, NIAID and NHLBI).

Integrins, cations and ligands: making the connection.

Summary.  Integrins are cell adhesion receptors that couple extracellular divalent cation‐dependent recognition events with intracellular mechanical and biochemical responses and vice versa, thus affecting every function of nucleated cells. The structural basis of this bidirectional signaling and its dependency on cations has been the focus of intensive study over the past three decades. Significant progress made recently in elucidating the three‐dimensional structure of the extracellular and cytoplasmic segments of integrins is giving valuable new insights into the tertiary and quaternary changes that underlie activation, ligand recognition and signaling by these receptors.

Structural basis for pure antagonism of integrin αVβ3 by a high affinity form of fibronectin

Integrins are important therapeutic targets. However, current RGD-based anti-integrin drugs are also partial agonists, inducing conformational changes that trigger potentially fatal immune reactions and paradoxical cell adhesion. Here we describe the first crystal structure of αVβ3 bound to a physiologic ligand, the tenth type III RGD domain of wild-type fibronectin (wtFN10), or to a high-affinity mutant (hFN10) shown here to act as a pure antagonist. Comparison of these structures revealed a central π-π interaction between Trp1496 in the RGD-containing loop of hFN10 and Tyr122 of the β3 subunit that blocked conformational changes triggered by wtFN10 and trapped hFN10-bound αVβ3 in an inactive conformation. Removing the Trp1496 or Tyr122 side chains or reorienting Trp1496 away from Tyr122 converted hFN10 into a partial agonist. These findings offer new insights into the mechanism of integrin activation and a basis for the design of RGD-based pure antagonists.

Characterization of a Conformationally Sensitive Murine Monoclonal Antibody Directed to the Metal Ion-Dependent Adhesion Site Face of Integrin CD11b

Integrin binding to physiologic ligands requires divalent cations and an inside-out-driven switch of the integrin to a high-affinity state. Divalent cations at the metal ion-dependent adhesion site (MIDAS) face of the α subunit-derived A domain provide a direct bridge between ligands and the integrin, and it has been proposed that activation dependency is caused by reorientation of the surrounding residues relative to the metal ion, forming an optimal binding interface. To gain more insight into the functional significance of the protein movements on the MIDAS face, we raised and characterized a murine mAb 107 directed against the MIDAS face of the A domain from integrin CD11b. We find that mAb 107 behaves as a ligand mimic. It binds in a divalent-cation-dependent manner to solvent-exposed residues on the MIDAS face of CD11b, blocks interaction of 11bA or the holoreceptor with ligands, and inhibits spreading and phagocytosis by human neutrophils. However, in contrast to physiologic ligands, mAb 107 preferentially binds to the inactive low-affinity form of the integrin, suggesting that its antagonistic effects are exerted in part by stabilizing the receptor in the low-affinity state. These data support a functional relevance of the protein movements on the MIDAS face and suggest that stabilizing the A domain in the low-affinity state may have therapeutic benefit.

Stable Coordination of the Inhibitory Ca2+ Ion at the Metal Ion-Dependent Adhesion Site in Integrin CD11b/CD18 by an Antibody-Derived Ligand Aspartate: Implications for Integrin Regulation and Structure-Based Drug Design

A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg2+ ion hexacoordinated at the metal ion-dependent adhesion site (MIDAS) in the integrin A domain. This interaction stabilizes the A domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking mAb 107 binds MIDAS of integrin CD11b/CD18 A domain (CD11bA), but in contrast, it favors the inhibitory Ca2+ ion over the Mg2+ ion at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of the Ca2+ ion at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca2+ ion. Binding of the Fab fragment of mAb 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that the denticity of the ligand Asp/Glu can modify the divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca2+ ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists.

Stable Coordination of the Inhibitory Ca 2+ Ion at the Metal Ion-Dependent Adhesion Site in Integrin CD11b/CD18 by an Antibody-Derived Ligand Aspartate: Implications for Integrin Regulation and Structure-Based Drug Design

A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg2+ ion hexacoordinated at the metal ion-dependent adhesion site (MIDAS) in the integrin A domain. This interaction stabilizes the A domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking mAb 107 binds MIDAS of integrin CD11b/CD18 A domain (CD11bA), but in contrast, it favors the inhibitory Ca2+ ion over the Mg2+ ion at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of the Ca2+ ion at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca2+ ion. Binding of the Fab fragment of mAb 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that the denticity of the ligand Asp/Glu can modify the divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca2+ ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists.

Atomic Basis for the Species-specific Inhibition of αV Integrins by Monoclonal Antibody 17E6 Is Revealed by the Crystal Structure of αVβ3 Ectodomain-17E6 Fab Complex

Background: 17E6, a primate-specific mouse mAb that inhibits αV integrins, is in phase II trials for treating cancer. Results: We determined crystal structure of the αVβ3–17E6 Fab complex, revealing the molecular basis of 17E6 specificity and function. Conclusion: 17E6 is an allosteric inhibitor of fibronectin-integrin interaction. Significance: The defined 17E6 epitope may help in developing novel therapeutics targeting related regions in other integrins. The function-blocking, non-RGD-containing, and primate-specific mouse monoclonal antibody 17E6 binds the αV subfamily of integrins. 17E6 is currently in phase II clinical trials for treating cancer. To elucidate the structural basis of recognition and the molecular mechanism of inhibition, we crystallized αVβ3 ectodomain in complex with the Fab fragment of 17E6. Protein crystals grew in presence of the activating cation Mn2+. The integrin in the complex and in solution assumed the genuflected conformation. 17E6 Fab bound exclusively to the Propeller domain of the αV subunit. At the core of αV-Fab interface were interactions involving Propeller residues Lys-203 and Gln-145, with the latter accounting for primate specificity. The Propeller residue Asp-150, which normally coordinates Arg of the ligand Arg-Gly-Asp motif, formed contacts with Arg-54 of the Fab that were expected to reduce soluble FN10 binding to cellular αVβ3 complexed with 17E6. This was confirmed in direct binding studies, suggesting that 17E6 is an allosteric inhibitor of αV integrins.