The Integrin Receptors: From Discovery to Structure to Medicines

Abstract

Innate immune cells perform vital tasks in detecting, seeking, and eliminating invading pathogens, thus ensuring host survival. However, loss of function of these cells or their overactive response to tissue injury often causes serious ailments. It is, therefore, crucial to understand at a basic level how these cells function in health and disease. A major step toward this goal came from studies I conducted in the late 1970s investigating the cause of life-threatening bacterial infections in a pediatric patient. This work led us to trace this disease to the inability of the patient's neutrophils to seek and clear infections due to an inherited deficiency in leukocyte adhesion caused by the loss of a plasma membrane glycoprotein complex now known as CD11/CD18 or β2 integrins. I followed this work by determining the 3-dimensional structures of integrins. These studies provided the foundation for understanding the unique properties of integrins in mediating bidirectional cell adhesion signaling and enabled a structure-guided design of compounds to dial down overactive integrins in common disorders, including thromboinflammatory and autoimmune diseases.

Keywords: cell adhesion; integrins; phagocytosis; structural biology.

Figure 1.. Defective Phagocytosis in a patient with recurrent bacterial infections.

a) Phagocytosis of serum-opsonized- or IgG-coated oil red O (ORO) particles by neutrophils from the patient (duplicate determinations) and controls (mean ± sd). b, c) DFP-treated and detergent-solubilized neutrophils fractionated on 7.5% SDS-PAGE and then stained with Coomassie (b) or PAS (c). d) Radioautograph of ¹²⁵I-surface-labeled neutrophils fractionated on 7.5% SDS-PAGE. Source: New Engl J Med vol 306 (12); March 25, 693–699. 1982.

Figure 2.. Tertiary changes in CD11bA domain upon ligand binding.

a) Ribbon diagrams of the superposed structures of liganded (light blue) and unliganded (gray) CD11bA domain. Ligand glutamate (E) induces an inward movement of strand A-loop- α1 helix, leading to the reorganization of strand F-loop-α7 helix that breaks the packing α7 helix, inclusive of I³¹⁶, against the hydrophobic central β-sheet and a 10Å descent of the C-terminal α7 helix. Major protein movements are indicated by arrows. Spheres represent the metal ions at MIDAS in their respective colors. b, c) The MIDAS motif in the liganded (light blue) (b) and unliganded (gray)(c) states. The metal ion at MIDAS is coordinated by residues from three surface loops, with carboxyl oxygen from ligand (E, ball, and stick) binding the metal ion monodentately, completing the metal octahedral coordinating sphere. In the unliganded state, an oxygen atom from a water molecule (ω3) replaces the ligand oxygen, and D²⁴² from the third surface loop moves in to coordinate the metal directly. Coordinating oxygen atoms are in red, and bonds are shown by dashed red lines. Water molecules are labeled ω1- ω3.

Figure 3.. Crystal structure of the αvβ3 ectodomain.

a) Ribbon diagrams of the ectodomain, related by 180° rotation along the x-axis, show the structure of the αv subunit (light blue) comprising four domains (labeled in the left panel), with metal ions at the base of the seven-bladed β-Propeller and at the α-genu shown as green spheres. The β3 subunit comprises 12 domains (labeled in the right panel), each in a different color for clarity. b) Ribbon tracings of the superposed βA domains of unliganded (gray)- and RGD-bound (orange) αvβ3 ectodomain with the metal ions shown in the respective colors. Bound ligand induces tertiary changes (arrows) also seen in αA domains (Fig. 2a) and in ADMIDAS metal ion coordination. The α1 helix and stand F-loop-α7 helix are thick and thinner tubes, respectively. See text for details.

Figure 4.. The ligand relay model.

a, b) Ribbon diagrams of the structure of the head segment (comprising the Propeller and βA domains) of an unliganded (a) and liganded (b) αA-containing integrin. The ligand (schematized by glutamate, E)-induced 10Å descent of the α7 helix of the αA domain allows invariant glutamate (E) at the bottom of the α7 helix to reach and ligate the metal ion at the βA MIDAS (cyan)(b), thus relaying the ligand occupancy state of αA to βA, which then undergoes similar tertiary changes (arrows) to those seen in the ligated αA domain (Fig. 2a).

Figure 5.. Cryo-EM structure of detergent-free full-length integrin αIIbβ3.

a) Cryo-EM map at 3.4 Å resolution of full-length platelet integrin αIIbβ3 is shown with ribbon diagrams for the αIIb chain (light blue) and β3 chain (orange-red). The αIIb and β3 transmembrane (TM) α-helices (labeled) are clearly visible in the map. The four αIIb domains and the α-genu are labeled. b) The cryo-EM density map is fitted with the bound γ-peptide from fibrinogen (atoms shown as spheres with carbons in green, nitrogens in blue, and oxygens in red). The structure is rotated 45^o counterclockwise in the membrane plane relative to that in (a). The distance measured between the MIDAS metal ion (cyan sphere) and αIIb’s Arg⁹⁶² at the extracellular leaflet of the membrane (solid vertical line) is 71.5 Å. The Calf1-Calf2 leg domain axis represented by a dotted dashed line linking metal ion at the αIIb genu (green sphere) to Ser⁹¹³ at the bottom of Calf2 is tilted from the normal by a 56° angle relative to the plane of the membrane (dotted line). c) The αIIbβ3 structure (yellow ribbon) in the EM density, oriented as in (a), has been fitted with a model of a fibrinogen dimer. The γ C-terminus of the fibrinogen crystal structure (3eio.pdb, shown in magenta) has been extended with an AlphaFold model (AlphaFold DB c9jeu5, shown in green) bound to the integrin ligand binding site. Inset, a closeup of the ligand binding site showing coordination of the Mg²⁺ ion at MIDAS (cyan sphere) with the fibrinogen ligand D⁴⁴⁴ (atoms shown as spheres with carbons in green and oxygens in red). Metal ions at ADMIDAS and LIMBS are in magenta and gray spheres, respectively. Source: Nat Commun. 2023. https://doi.org/10.1038/s41467-023-39763-0

Figure 6.. Drug-induced quaternary changes in full-length αIIbβ3.

a) Ribbon diagram of the unliganded headpiece of the cryo-EM structure of full-length αIIbβ3 shown in Fig. 5a (αIIb in light blue and β3 in gray). The metal ions (spheres) at LIMBS (gray), MIDAS (cyan), and ADMIDAS (magenta) of the βA domain are shown. b) Ribbon diagram of the ligand-mimetic drug tirofiban (atoms shown as spheres with carbons in green, nitrogens in blue, oxygens in red, and sulfurs in yellow) bound to the αIIbβ3 headpiece of the full-length αIIbβ3 structure. Only the integrin head plus the Hybrid, PSI, and EGF1 domains are clearly visible in the map. The ligand-induced protein movements in βA (arrows) result in a swingout (arrow) of the Hybrid domain, associated with invisible leg and TM domains. c) Ribbon tracings of the superposed βA domains of unliganded (gray)- and tirofiban-bound (orange) full-length αIIbβ3. Tirofiban engages the Propeller via D²²⁴ and the MIDAS metal ion and induces the classical tertiary changes in the βA domain: the inward movement of the α1 helix brings S¹²³ and ADMIDAS closer to the MIDAS metal ion and rearrangements of the stand F-loop-α7 helix forces the α7 helix downward. Helices are loops are shown as thick and thin tubes, respectively.

Figure 7.. Structures of integrins in complex with pure integrin inhibitors.

a) Ribbon diagrams of the superposed inactive (gray) and mAb107-bound (light blue) crystal structures of the αA domain from integrin CD11b. The mAb107-bound αA domain is stabilized in the inactive conformation via the amphipathic α7 helix packing against the hydrophobic protein core. b) Closeup view of the MIDAS region (encircled in (a) of the mAb107-bound structure showing how the αA domain is kept inactive. Bidentate coordination of the MIDAS metal ion by the ligand Asp (D¹⁰⁷) stabilizes the inhibitory Ca²⁺ at MIDAS. c) Ribbon diagrams of superposed βA domains of unliganded (gray)- and m-tirofiban-bound (orange) cryo-EM structures of full-length αIIbβ3. Bound m-tirofiban keeps the βA domain in the unliganded conformation with the α7 helix packed, including L³⁴³, against the central β-sheet. d) Closeup view of the MIDAS region of m-tirofiban-bound βA domain showing how βA is kept inactive. m-tirofiban prevents the inward movement of the strand A-loop-α1 helix towards MIDAS via respective π- π and cation-π interactions of the benzoxazole moiety with Y and R²¹⁴ (not shown). This maintains the ADMIDAS metal ion-mediated link between the α1 and α7 helices and the indirect coordination of the MIDAS Mg²⁺ by S¹²³ via a water molecule (ω1).