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Crystal Structure of the Extracellular Segment of Integrin αVβ3 in Complex with an Arg-Gly-Asp Ligand

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Science  05 Apr 2002:
Vol. 296, Issue 5565, pp. 151-155
DOI: 10.1126/science.1069040

Abstract

The structural basis for the divalent cation–dependent binding of heterodimeric αβ integrins to their ligands, which contain the prototypical Arg-Gly-Asp sequence, is unknown. Interaction with ligands triggers tertiary and quaternary structural rearrangements in integrins that are needed for cell signaling. Here we report the crystal structure of the extracellular segment of integrin αVβ3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence. The ligand binds at the major interface between the αV and β3 subunits and makes extensive contacts with both. Both tertiary and quaternary changes are observed in the presence of ligand. The tertiary rearrangements take place in βA, the ligand-binding domain of β3; in the complex, βA acquires two cations, one of which contacts the ligand Asp directly and the other stabilizes the ligand-binding surface. Ligand binding induces small changes in the orientation of αV relative to β3.

Integrins are adhesion receptors that mediate vital bidirectional signals during morphogenesis, tissue remodeling, and repair [reviewed in (1)]. These heterodimers are formed by the noncovalent association of an α and a β subunit, both type I membrane proteins with large extracellular segments. In mammals, 18 α and 8 β subunits assemble into 24 different receptors. Integrins depend on divalent cations to bind their extracellular ligands. Although these ligands are structurally diverse, they all use an acidic residue during integrin recognition. Specificity for a particular ligand is then determined by additional contacts with the integrin. High affinity binding of integrins to ligands is usually not constitutive but is elicited in response to cell “activation” signals (so-called “inside-out” signaling) that alter the tertiary and quaternary structure of the extracellular region, making the integrin ligand-competent. Ligand binding, in turn, induces structural rearrangements in integrins that trigger “outside-in” signaling [reviewed in (2)].

Integrins are grouped into two classes based on the presence or absence of an extracellular ∼180 amino acid A-type domain (αA) (3). In the nine αA-containing integrins (αA-integrins), αA is necessary and sufficient for the divalent cation–dependent binding to physiologic ligands (3). The structures of isolated αA domains in “liganded” and “unliganded” conformations (4–8) have revealed how this domain interacts with ligands. A metal ion is coordinated at the ligand-binding interface of αA through a conserved five amino acid motif, the metal ion–dependent adhesion site (MIDAS), and the metal coordination is completed by a glutamate from the ligand (4,6) or, in its absence, by a water molecule (9). In αA-lacking integrins, ligand recognition requires an αA-like domain (βA) present in all integrin β subunits (4, 10).

The crystal structure of the extracellular segment of the αA-lacking integrin αVβ3 was previously determined in the presence of Ca2+ (αVβ3-Ca) (10). It consists of 12 domains assembled into an ovoid head and two “legs.” The putative ligand-binding head is primarily formed of a seven-bladed β-propeller domain from αV and a βA domain from β3. These two domains resemble the Gβ and Gα subunits of G-proteins, respectively, and contact each other in a strikingly similar manner (10). We now report the structure of extracellular αVβ3 in complex with the cyclic pentapeptide ligand Arg-Gly-Asp-{d-Phe}-{N-methyl-Val-}, called cyclo(RGDF=N{Me}V [P5 in (11)], and in the presence of the proadhesive cation Mn2+, αVβ3-RGD-Mn (Table 1). We have also determined the structure of the unliganded receptor in the presence of Mn2+ (αVβ3-Mn) for comparison (Table 1). The two structures contain the previously reported extracellular residues of the integrin (10). αVβ3-Mn contains six Mn2+ions (replacing each of the six Ca2+ ions in αVβ3-Ca), and αVβ3-RGD-Mn contains the cyclic pentapeptide plus eight Mn2+ ions. Replacement of Ca2+ with Mn2+ at all six sites in the αVβ3-Mn structure did not result in important structural rearrangements in the integrin. As with αVβ3-Ca (10), no metal ion is visible at MIDAS in αVβ3-Mn. Figure 1 shows representative electron density maps (Fig. 1, A through D) and a ribbon diagram (Fig. 1E) of the integrin-pentapeptide complex.

Figure 1

Structure of αVβ3-Mn complexed with cyclo(RGDf-N{Me}V). (A) Stereoview of a 2F oF c electron density map of the peptide-integrin complex, where o and c are the observed and calculated structures, respectively. Cutoff is 5.0σ for Mn2+ and 1.0σ for ligand residues. Densities (magenta) of the adjacent metal ions at ADMIDAS, MIDAS, and LIMBS (shown here and in subsequent figures as violet, cyan, and gray respectively) are from the same map. (B) A Fourier anomalous difference map in the same region as in (A) of αVβ3-Mn; only the density (cyan) of the Mn2+ ion at ADMIDAS is detected. (C andD) A Fourier anomalous difference map showing the densities (cyan) of the Mn2+ ions (orange), four in β hairpin loops of blades 4 to 7 of the propeller (C) and one at the αV genu (D) in the peptide-integrin complex. (E) Ribbon drawing (20) of the αVβ3-RGD-Mn structure. In this and subsequent figures, αV and β3 are shown in blue and red, respectively. The peptide is bound at the propeller-βA domain interface with the ADMIDAS, MIDAS, and LIMBS metal ions shown. The carbon, nitrogen, and oxygen atoms of cyclo(RGDf-N{Me}V) are shown in yellow, blue, and red, respectively.

Table 1

Data collection and refinement statistics [Supplemental note 1, (14)]. Diffraction data were sharpened with a B factor of –40.0Å2. The αVβ3-Mn and αVβ3-RGD-Mn structures were solved by molecular replacement at 3.3 Å and 3.2 Å resolution, respectively, using the previously reported αVβ3-Ca structure as the initial model. For the αVβ3-Mn structure, the original coordinates were modified by removing all six calcium ions. Coordinates were then subjected to rigid body minimization. A F oF cdifference map showed positive density in all six previously determined calcium-binding sites. The eight metal ion densities in the structure were all assigned as manganese because crystals were soaked with buffer containing 5 mM MnCl2. The positions of manganese ions were confirmed from the anomalous difference Fourier maps using data collected at the wavelength 1.2398 Å, where Mn2+ has reasonable anomalous contribution (f" = 1.96 electrons). For the αVβ3-RGD-Mn structure, a similar F oF c difference density map showed clear density for all five amino acids of the cyclic peptide ligand and for eight Mn2+ ions: six at the original sites and two in the vicinity of the ligand. Each model was then modified according to the difference density features and refined using bulk solvent correction and several rounds of simulated annealing protocols in XPLOR (22). About 5% of reflections were used to calculate the free R factor in each case. The reflections included in the two ”free sets“ were the same as those used for the αVβ3-Ca structure determination. The electron density maps calculated with the three independent data sets (for αVβ3-Ca, αVβ3-Mn, and αVβ3-RGD-Mn) do not allow us to trace the PSI, EGF-1, and EGF-2 domains. In addition, these three density maps show some variability in the NH2-terminal part of EGF-3, leaving open the possibility that alternative disulphide bridges in this region exist.

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The αVβ3-RGD-Mn structure reveals that the pentagonal peptide inserts into a crevice between the propeller and βA domains on the integrin head (Fig. 1E). The Arg-Gly-Asp, or RGD, sequence makes the main contact area with the integrin, and each residue participates extensively in the interaction, which buries 355 Å2 or 45% of the total surface area of the peptide. The Arg and Asp side chains point in opposite directions, exclusively contacting the propeller and βA domains, respectively. The five Cα atoms of the cyclic peptide form a slightly distorted pentagon. Molecular dynamics simulations of the peptide in the absence of the integrin, performed using the same geometric parameters used for the crystallographic refinement, result in a more regular pentagonal shape with roughly equal inter–Cα atom distances (data not shown). Thus, distortion of the peptide ring is apparently related to contact with αVβ3-Mn. The main chain conformation of the RGD motif in the pentapeptide is almost identical to that of the RGD tripeptide in the natural ligand Echistatin (12, 13), suggesting that the structure presented here can serve as a basis for understanding the interaction of integrins with other and larger RGD-containing ligands.

The Arg side chain inserts into a narrow groove at the top of the propeller domain (Fig. 2A), formed primarily by the D3-A3 and D4-A4 loops. The arginine guanidinium group is held in place by a bidentate salt bridge to Asp218 at the bottom of the groove and by an additional salt bridge to Asp150 at the rear (Fig. 2B). The contacts leave most of the upper portion of the Arg side chain exposed to solvent, whereas the spacious rear of the groove probably contains water molecules that may provide additional contacts to the Arg guanidinium group.

Figure 2

The ligand-integrin binding site. (A) Surface representation of the ligand-binding site, with the ligand peptide shown as ball-and-stick model. Color code for the ligand and the two visible Mn2+ ions (MIDAS and ADMIDAS) is as in Fig. 1. (B) Interactions between ligand and integrin. The peptide (yellow) and residues interacting with the ligand or with Mn2+ ions are shown in ball-and-stick representation. αV and β3 residues are labeled blue and red, respectively. Oxygen and nitrogen atoms are in red and blue, respectively. The three Mn2+ ions in β3 at MIDAS, ADMIDAS, and LIMBS are also shown. Hydrogen bonds and salt bridges (distance cutoff, 3.5 Å) are represented with dotted lines.

Contacts between the ligand Asp and βA primarily involve the Asp carboxylate group, which protrudes into a cleft between the βA loops A′-α1 and C′-α3 and forms the center of an extensive network of polar interactions (Fig. 2B). One of the Asp carboxylate oxygens contacts a Mn2+ ion at MIDAS in βA (Fig. 2B). The second Asp carboxyl oxygen forms hydrogen bonds with the backbone amides of Tyr122 and Asn215 and also contacts the aliphatic portion of the Arg214 side chain. Additional contacts involve the hydrophobic portion of the Asp side chain and the beta carbon atom of Asn215. Unlike the ligand Arg, the ligand Asp side chain is completely buried in the complex.

The glycine residue, which completes the prototype RGD ligand sequence, lies at the interface between the α and β subunits (Fig. 2B). It makes several hydrophobic interactions with αV, the most critical of which appears to be the contact with the carbonyl oxygen of Arg216. The remaining two residues of the pentapeptide face away from the αβ interface and are not in the consensus ligand sequence.

The peptidyl aspartate contacts βA in a manner that strikingly resembles the interaction of αA with its ligands (4,6) (Fig. 3); in both cases, an acidic ligand residue coordinates the receptor via a metal ion in MIDAS. However, βA differs from αA in that the latter can bind a metal ion in MIDAS even in its unliganded state (5,9). The one difference between the two sites is the replacement of a conserved Thr, which contacts the cation in liganded αA, with Glu220 in βA. In the unliganded αVβ3-Mn structure, the Glu220 side chain intrudes into the MIDAS site, approaching the space where a cation would bind. Thus, it appears to reduce the affinity for cations at MIDAS through steric hindrance. In the liganded αVβ3-RGD-Mn structure, the Glu220 side chain occupies a different position, allowing accommodation of a cation at MIDAS.

Figure 3

Diagram of the MIDAS motif in βA (A and B) and αA from CD11b (C andD). (A) and (B) MIDAS residues (single letter abbreviations: S, Ser; E, Glu; D, Asp; T, Thr) in unliganded (A) and liganded (B) βA. Coordinating side chains are shown in ball-and-stick representations with oxygen atoms in red, carbon in green; the ligand aspartate is in gold. In addition to the ligand aspartate, the Mn2+ (cyan) in the βA MIDAS is coordinated directly with the hydroxyl oxygens of Ser121 and Ser123 and with one carboxylate oxygen from Glu220. The carboxyl oxygens of Asp119 and Asp251 of βA lie within 6Å of the metal ion and likely mediate additional contacts through water molecules similar to the liganded forms of αA (D). The Mn2+ ion at ADMIDAS (magenta) is present in (A) and (B). The Mn2+ ions at MIDAS and at LIMBS (cyan and gray, respectively) are only present in (B). (C) and (D) MIDAS residues in unliganded (C) and liganded (D) αA from CD11b. The metal ion (cyan) is present in both. Water molecules are labeled ω; the pseudoligand glutamate is in gold. Hydrogen bonds and metal ion coordination are represented with dotted yellow lines.

In addition to incorporating Mn2+ at MIDAS when liganded, βA also unexpectedly incorporates a second Mn2+ion. Only 6 Å from MIDAS, this ion defines a ligand-associated metal binding site (LIMBS) formed by the other carboxylate oxygen of Glu220; the side chains of Asp158, Asn215, and Asp217; and the carbonyl oxygens of Asp217 and Pro219 (Fig. 2B). Although the LIMBS Mn2+ ion does not contact the ligand, coordination of Mn2+ nevertheless depends on it. Asp158 and Glu220 occupy different positions in the unliganded structure, and, therefore, the coordination sphere for LIMBS does not exist. The most likely role of LIMBS is to stabilize the reoriented Glu220 and to add conformational stability and structural rigidity to the ligand-binding surface. Taken together, the above data explain the structural basis for conservation of the RGD consensus in integrin ligands and for certain loss-of-function disease mutations in β3 integrins [Supplemental note 2 (21)].

Binding of the pentapeptide ligand is associated with tertiary and quaternary changes in αVβ3-Mn. Changes in tertiary structure involve βA, affecting primarily its α1-α2 loops and helices and the α2-C', F-α7, and B-C (“ligand-specificity”) loops (Fig. 4, A and B). The observed movements appear to be causally linked to the top of helix α1 which approaches MIDAS, permitting contacts with both MIDAS cation and ligand through Ser121, Tyr122, and Ser123. In the complex, the backbone amide and carbonyl oxygens of Tyr122directly contact the ligand Asp, and both serine side chains coordinate the MIDAS cation. Thus, α1 is fastened to the ligand-MIDAS assembly within the complex. The ADMIDAS (adjacent to MIDAS) cation moves in concert with α1 because it is primarily coordinated by α1 residues Asp126 and Asp127; this changes its coordination sphere slightly from that of the unliganded structure (10) (only its coordination by the carbonyl oxygen of Met335 is replaced by a carboxylate oxygen from Asp251). Most of the remaining structural changes can be viewed as indirectly caused by the shift of α1: α1′ directly follows α1 in sequence, and α2 and the top of α7 flank α1′. The ligand-specificity region also approaches the ligand. This movement may be related to a salt bridge in this region between Asp179and Arg214. Arg214 is near the ligand Asp, and it does not form a salt bridge to Asp179 in the unliganded structure. The functional implications of these changes are reflected by the location in the α1-α2 segment of βA of epitopes both for activation and inhibitory monoclonal antibodies (15–18).

Figure 4

Ligand-induced structural changes in βA in comparison with those of αA (from CD11b). (A) Superposition, in stereo, of the αVβ3-Mn (gray) and αVβ3-RGD-Mn (red) structures. The superposition is based on the Cα atoms of the central β-sheet [43 atoms per structure; root mean square deviation (RMSD), 0.42 Å]. Residues of αVβ3-RGD-Mn with a distance of more than 1.5 Å to corresponding residues of αVβ3-Mn are shown with thicker red lines. The major structural changes in βA involve helices α1, α1′, α2, the F-α7 loop, and the ligand-specificity region. (B) Magnified view of the rearrangements at the ligand-binding site in βA. Superposition of the propeller and βA domains of αVβ3-Mn (gray) and αVβ3-RGD-Mn (αV, blue; β3, red) is based on the Cα atoms of the αV propeller domain. The directions of protein movements (including the 4 Å displacement of Mn2+ at ADMIDAS) are indicated by red arrows. This view differs from (A) by a rotation of 180° around a vertical axis. (C) Superposition, in stereo, of the “liganded” (red) and “unliganded” forms of αA from the CD11b integrin. The metal ion sphere at MIDAS is in cyan. The superposition is based on the Cα atoms of the central β-sheet (43 atoms; RMSD = 0.43 Å). Residues of liganded αA with a distance of more than 1.5 Å to corresponding residues of unliganded αA are shown with thicker red lines. The major structural changes in αA involve helices α1, α7, the F-α7, and E-α6 loops. Arrows (red) indicate the direction of the major protein movements in each case.

The above tertiary changes observed in the liganded form of βA resemble those seen in liganded αA (6,8, 9) (Fig. 4). In αA, a major distinguishing feature of its transition from the unliganded to the liganded state is a 10 Å downward shift of the COOH-terminal α7 helix with realignment of its hydrophobic contacts (Fig. 4C) (6, 8,9). However, the position of the α7 helix in liganded βA does not change (it already occupies an equivalent position to liganded αA when ligand is absent). One likely interpretation of these data is that activation (ligand-competency) in αA and βA is achieved by different mechanisms. Reorientation of the COOH-terminal α7 helix, perhaps in response to inside-out signaling, makes αA ligand-competent in an allosteric manner (8). In βA, where such movement of the α7 helix is less likely, reorientation of the MIDAS Glu220 residue (which is invariant in βA but not αA domains), results in a ligand-competent form by unblocking MIDAS. A second interpretation of these data is that the conformation of βA in the unliganded αVβ3-Mn and αVβ3-Ca structures represents a ligand-competent state of the A-type domain, captured in the context of an integrin heterodimer. In this scenario, the tertiary changes observed here in βA are ligand-induced.

Quaternary rearrangements in the integrin head region are also observed in the complex. The interface between βA and the αV propeller undergoes a small change, with the two domains moving closer together at the peptide-binding site [see animated Supplemental fig. 1, A and B (19)]. In addition, the propeller undergoes a small rotation at the propeller-thigh interface, with βA moving in concert [see animated Supplemental fig. 1, A and B (19)]. Thus, as in the case of G-proteins, ligand binding to βA alters its orientation relative to the propeller. It is also remarkable that both tertiary and quaternary changes are observed in an integrin in the presence of its smallest recognition unit, even within the constrained crystal lattice. Natural integrin ligands are significantly larger, structurally diverse and often multivalent. Thus, the present conformational rearrangements likely represent a minimalist view of the scope of changes in the receptor that take place during integrin-ligand interactions.

  • * These two authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: arnaout{at}receptor.mgh.harvard.edu

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