• Conformational extension is essential for integrin to fulfill its cell-adhesion function in platelet-mediated hemostasis and thrombosis.

  • High-resolution structures of β3 integrin in intermediate and extended conformations reveal atomic-level conformational rearrangements.

Cells use adhesion receptor integrins to communicate with their surroundings. Integrin activation and cellular signaling are coupled with change from bent to extended conformation. β3 integrins, including αIIbβ3, which is essential for the function of platelets in hemostasis and thrombosis, and αVβ3, which plays multiple roles in diverse cell types, have been prototypes in understanding integrin structure and function. Despite extensive structural studies, a high-resolution integrin structure in an extended conformation remains to be determined. The human β3 Leu33Pro polymorphism, located at the PSI domain, defines human platelet-specific alloantigens 1a and 1b (HPA-1a/b), immune response to which is a cause of posttransfusion purpura and fetal/neonatal alloimmune thrombocytopenia. Leu33Pro substitution has also been suggested to be a risk factor for thrombosis. Here we report the crystal structure of the β3 headpiece in either Leu33 or Pro33 form, both of which reveal intermediate and fully extended conformations coexisting in 1 crystal. These were used to build high-resolution structures of full-length β3 integrin in the intermediate and fully extended states, agreeing well with the corresponding conformations observed by electron microscopy. Our structures reveal how β3 integrin becomes extended at its β-knee region and how the flexibility of β-leg domains is determined. In addition, our structures reveal conformational changes of the PSI and I-EGF1 domains upon β3 extension, which may affect the binding of conformation-dependent anti–HPA-1a alloantibodies. Our structural and functional data show that Leu33Pro substitution does not directly alter the conformation or ligand binding of β3 integrin.

Integrins are a family of 24 α/β heterodimeric adhesion receptors that transmit bidirectional signals across the cell membrane.1  Each integrin subunit contains a large extracellular domain composed of a headpiece and leg piece, a transmembrane domain, and usually a short cytoplasmic tail (supplemental Figure 1, available on the Blood Web site).2  The α headpiece consists of β-propeller and thigh domains and an inserted αI domain on top of the β-propeller in a subset of α subunits, such as leukocyte-specific αL. The α leg piece includes calf-1 and ‒2 domains. The β headpiece contains βI, hybrid, PSI, and I-EGF1 domains (Figure 1A). The β leg piece contains I-EGF2 to -4 and β-tail domains (Figure 1A). Structural studies in the last 17 years, largely based on β3 integrins, including αIIbβ3 and αVβ3,3-11  have greatly advanced our understanding of integrin conformational regulation upon activation. Platelet-specific αIIbβ3 plays an essential role in hemostasis and thrombosis,12  and αVβ3 is important for the growth and survival of many cell types.13,14  In the resting state, integrin adopts a bent conformation, with the headpiece folded onto the leg piece (supplemental Figure 1A). Crystallographic and electron microscopy (EM) studies of αIIbβ3 and αVβ3 have revealed 2 major conformational rearrangements: the swing-out motion of the hybrid domain, resulting in headpiece opening, and the switchblade-like movement of the headpiece from the leg domains, leading to integrin extension and leg separation (supplemental Figure 1B-C).2,7  Despite the visualization of β3 extension by EM at low resolution (supplemental Figure 1B-C), a high-resolution extended β3 structure is not available.

Figure 1.

Overall structure of the single-chain chimeric β3headpiece. (A) Diagram of the native and chimeric β3 (cβ3) constructs. The cβ3-E2 construct was truncated at the I-EGF2 domain. (B) The 2 copies of cβ3-E2 structure in the asymmetric unit. N-linked glycans are shown as sticks with green carbons, red oxygens, and blue nitrogens. Metal ions are shown as spheres. Disulfide bonds are blue sticks. (C) Structural alignment of the 2 cβ3-E2 molecules in the asymmetric unit. (D) Structural comparison of the extended cβ3-E2 structure (in red; disulfide bonds in blue) with the native β3 structure (PDB 3FCS; in cyan; disulfide bonds in yellow). The 2 structures were aligned on the hybrid domain.

Figure 1.

Overall structure of the single-chain chimeric β3headpiece. (A) Diagram of the native and chimeric β3 (cβ3) constructs. The cβ3-E2 construct was truncated at the I-EGF2 domain. (B) The 2 copies of cβ3-E2 structure in the asymmetric unit. N-linked glycans are shown as sticks with green carbons, red oxygens, and blue nitrogens. Metal ions are shown as spheres. Disulfide bonds are blue sticks. (C) Structural alignment of the 2 cβ3-E2 molecules in the asymmetric unit. (D) Structural comparison of the extended cβ3-E2 structure (in red; disulfide bonds in blue) with the native β3 structure (PDB 3FCS; in cyan; disulfide bonds in yellow). The 2 structures were aligned on the hybrid domain.

Close modal

A Leu33Pro polymorphism at the β3 PSI domain defines the human platelet-specific alloantigen 1 (HPA-1) system.15  Mismatched HPA-1a/b (Leu33/Pro33) is a major cause of 2 severe bleeding disorders: fetal/neonatal alloimmune thrombocytopenia and posttransfusion purpura, which result from the generation of anti–HPA-1a alloantibodies.16,17  In addition, whether the Pro33 variant enhances β3 activity, as a risk factor for thrombosis, is controversial.18-20  It is unknown whether Leu33Pro substitution affects the conformation of the PSI domain, which undergoes outward movement along with headpiece opening and extension.

Crystallographic studies of a full-length extended integrin have not been successful. This is largely due to the flexibility of the β-leg domains in the extended conformation as indicated by EM studies.2  In our study, to obtain the high-resolution crystal structure of an extended β3 integrin, we engineered a cβ3 fragment containing the headpiece and β-knee region at where β3 becomes extended. cβ3 could be expressed without an α subunit. We solved the crystal structures of the cβ3 fragment in both Pro33 and Leu33 forms. The structures revealed intermediate and fully extended β3 conformations at the β knee, providing high-resolution information on how β3 becomes extended. The structures of full-length β3 integrin were built in both intermediate and fully extended conformations, agreeing well with the corresponding conformations observed by EM. Our structural and functional studies provide an atomic-level understanding of integrin conformational regulation on the cell surface.

DNA constructs

The αIIb, αV, αL, α5, β1, β2, β3, and EGFP-talin head constructs were as described previously.21-23  Mutations were generated by the QuikChange XL Site-Directed Mutagenesis Kit (Agilent Technologies). The cβ3 construct was designed by replacing the β3 βI domain (residues Tyr110-Gly349) with the αL αI domain (residues Gly128-Lys304). The full-length cβ3 DNA construct was generated by overlapping polymerase chain reaction and cloned into the pcDNA3.1/Myc-His+ A vector using HindIII and XbaI restriction sites. The DNA fragment of the cβ3 headpiece containing the PSI with either Pro33 or Leu33, hybrid, αI, I-EGF1, and I-EGF2 domains was cloned into the XhoI and MluI sites of the ExpressTag 1 vector, which was based on the pIRES2-EGFP vector.24  The construct was C-terminal tagged with an HRV 3C protease cleavage site, protein C epitope, hexahistidine, and streptavidin binding protein affinity tag. All constructs were validated by DNA sequencing.

Protein expression and purification

The cβ3 headpiece construct was transfected into HEK293S GnTI cells. The transfected cells were cultured in a medium containing 1 mg/mL of G418 for at least 10 days before a pool of EGFP+ cells were sorted. The EGFP+ cells were cultured in roller bottles in the selection medium. The cell-culture supernatant was collected every 3 to 5 days. The cβ3 headpiece proteins were purified from the supernatant using Strep-Tactin Superflow Plus beads following the manufacturer’s protocol (Qiagen). The proteins were treated with HRV 3C protease to remove the tags and then with Endoglycosidase H to remove the high-mannose type of N-linked glycans. The untagged proteins were finally purified with a Superdex 200 column (GE Healthcare) equilibrated with a buffer containing 20 mM of Tris-hydrochloride (potential of hydrogen [pH], 7.5), 150 mM of sodium chloride (NaCl), 1 mM of calcium chloride, and 1 mM of magnesium chloride and concentrated to 10 mg/mL for protein crystallization.

Protein crystallization and crystallography

Crystallization was performed at 4°C by hanging-drop vapor diffusion method with 1:1 volume/volume ratio of protein and cocktail solutions. Both Pro33 and Leu33 forms of cβ3 headpiece proteins were crystallized in either 20% PEG 6000 plus 0.1 M of Tris-hydrochloride (pH, 8.5) or 12% PEG 8000 plus 0.2 M of NaCl and 0.1 M of N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (pH, 7.5). The crystals were cryoprotected with additional glycerol in the 18% PEG 8000 solution in 5% increments up to 20% final concentration. For dehydration, crystals were soaked in the 18% PEG 8000 solution containing 2 M of NaCl and 20% glycerol at 4°C for >3 days before being flash frozen in a pool of liquid nitrogen.

Crystal X-ray diffraction data were collected at the APS LS-CAT beamline 21-ID-F and processed with iMosflm.25  The structure was solved by molecular replacement with PHASER26  using the αL αI domain crystal structure (PDB 3F74) as a search model. The structures were manually built in Coot27  and refined using PHENIX.REFINE.28  The atomic coordinates and structural factors have been deposited in the protein data bank under accession numbers 6BXF (for β3-Leu33), 6BXB (for β3-Pro33 after dehydration), and 6CKB (for β3-Pro33 before dehydration).

Ligand and conformation-specific antibody binding assay

The ligand mimetic PAC-1 (BD Biosciences), human fibronectin (Sigma-Aldrich) labeled with Alexa Fluor-647, and human ICAM-1 with a C-terminal Fc tag (expressed as a secreted protein in HEK293FT cells) were used as ligands for αIIbβ3, αVβ3, and αLβ2, respectively. 9EG7 (BD Biosciences) is a rat anti-β1 mAb that reports β1 integrin extension.29,30  Both Alexa Fluor-647–labeled fibronectin and 9EG7 were used for measuring the activation of α5β1 integrin. The ligand or monoclonal antibody (mAb) binding assay using integrin-transfected HEK293FT cells was performed as described previously in the presence of integrin inhibitor Ca2+/Mg2+ (1 mM) or Ca2+ (0.2 mM) plus Mn2+ (2 mM).21-23  The binding of ligand or conformation-specific mAb was presented as the mean fluorescence intensity normalized to the mean fluorescence intensity of integrin expression reported by the mAbs AP3, TS2/4, and MAR4 for β3, αLβ2, and α5β1, respectively.

Design of a single-chain cβ3 headpiece construct

The βI and αI domains are structural homologs.31  The αI but not βI domains can be expressed autonomously.32-34  By replacing the βI with the αI domain of αL integrin, the cβ subunit could be expressed as a single-chain construct independent of an α subunit (A.M.M.T., Z.W., D.Z., Y.Z., Brian R. Curtis, and J.Z., manuscript submitted April 2018). We solved the crystal structure of the single-chain cβ3 ectodomain that adopts a bent conformation resembling β3 in the αIIbβ3 or αVβ3 complex (A.M.M.T., Z.W., D.Z., Y.Z., Brian R. Curtis, and J.Z., manuscript submitted April 2018). In the bent conformation of the β3 subunit, the headpiece folds onto the lower leg domains, forming close contacts between the hybrid and I-EGF3 to -4 domains (supplemental Figure 1A).4,5  Such interactions help maintain β3 in the bent conformation even in the absence of an α subunit (A.M.M.T., Z.W., D.Z., Y.Z., Brian R. Curtis, and J.Z., manuscript submitted April 2018) and upon disruption may facilitate integrin extension. The β integrin extension occurs at the I-EGF1/2 junction, denoted as β knee (supplemental Figure 1B-C; Figure 1A).2  We generated a cβ3 headpiece construct containing the PSI, hybrid, αI, I-EGF1, and I-EGF2 domains, namely cβ3-E2 (Figure 1A). In the absence of the restrains of the lower leg domains, we expected cβ3-E2 might adopt an extended conformation. To investigate whether the Leu33Pro polymorphism affects the β3 conformation, both Leu33 and Pro33 forms of cβ3-E2 were prepared.

Two conformations of a single-chain cβ3 headpiece coexist in 1 crystal

Both Leu33 and Pro33 forms of cβ3-E2 crystals belong to the same space group (Table 1). The original crystals diffracted X-ray up to 2.8-Å resolution, which was improved by crystal dehydration. The unit cell was shortened in 1 dimension after dehydration (Table 1), as a result of more tightly packed crystal lattice (supplemental Figure 2A). The cβ3-E2-Pro33 structure obtained after crystal dehydration was refined to a resolution of 2.39 Å, with an Rfree of 0.267. The cβ3-E2-Pro33 structure obtained without crystal dehydration was refined to a resolution of 2.80 Å, with an Rfree of 0.296. The cβ3-E2-Leu33 structure was obtained with the crystals without dehydration and was refined to a resolution of 3.2 Å, with an Rfree of 0.335 (Table 1). Two cβ3-E2 molecules are present per asymmetric unit (Figure 1B).

Table 1.

Statistics of X-ray diffraction data and structure refinement

Data collection3-E2-P333-E2-L33
Before dehydrationAfter dehydrationNo dehydration
Space group P21 P21 P21 
Unit cell    
a, b, c, Å 59.08, 80.91, 126.72 59.56, 79.84, 116.16 59.07, 81.47, 127.96 
 α, β, γ, ° 90.0, 96.4, 90.0 90.0, 91.42, 90.0 90.0, 97.1, 90.0 
Wavelength, Å 0.97872 0.97872 0.97872 
Resolution, Å 2.80 2.39 3.20 
Rmerge, %* 15.0 (>100) 13.8 (>100) 22.6 (>100) 
No. of reflections, measured/unique 222 851/29 474 328 778/43 236 152 292/20 112 
I/σ(I)* 8.6 (0.5) 9.6 (0.7) 6.5 (0.6) 
Completeness, %* 100 (100) 99.7 (98.8) 100 (100) 
Redundancy* 7.6 (7.6) 7.6 (6.8) 7.6 (7.6) 
CC1/2* 0.995 (0.118) 0.996 (0.121) 0.959 (0.134) 
Refinement    
 Resolution, Å 2.80 2.39 3.20 
 Unique reflections, work/free 29 394/1 479 43 143/2 128 20 063/1 019 
 Rwork/Rfree 0.242/0.296 0.219/0.267 0.265/0.335 
 No. of atoms, protein/carbohydrates/water 7 223/70/4 7 215/70/170 7 197/98/8 
 Molecules per asymmetric unit 
 RMSD from ideal    
  Bond lengths, Å 0.009 0.004 0.003 
  Bond angles, ° 1.052 0.650 0.625 
 Ramachandran plot, favored/allowed/outliers, % 87.57/10.81/1.62 92.64/7.14/0.22 87.87/9.75/2.38 
 PDB code 6CKB 6BXB 6BXF 
Data collection3-E2-P333-E2-L33
Before dehydrationAfter dehydrationNo dehydration
Space group P21 P21 P21 
Unit cell    
a, b, c, Å 59.08, 80.91, 126.72 59.56, 79.84, 116.16 59.07, 81.47, 127.96 
 α, β, γ, ° 90.0, 96.4, 90.0 90.0, 91.42, 90.0 90.0, 97.1, 90.0 
Wavelength, Å 0.97872 0.97872 0.97872 
Resolution, Å 2.80 2.39 3.20 
Rmerge, %* 15.0 (>100) 13.8 (>100) 22.6 (>100) 
No. of reflections, measured/unique 222 851/29 474 328 778/43 236 152 292/20 112 
I/σ(I)* 8.6 (0.5) 9.6 (0.7) 6.5 (0.6) 
Completeness, %* 100 (100) 99.7 (98.8) 100 (100) 
Redundancy* 7.6 (7.6) 7.6 (6.8) 7.6 (7.6) 
CC1/2* 0.995 (0.118) 0.996 (0.121) 0.959 (0.134) 
Refinement    
 Resolution, Å 2.80 2.39 3.20 
 Unique reflections, work/free 29 394/1 479 43 143/2 128 20 063/1 019 
 Rwork/Rfree 0.242/0.296 0.219/0.267 0.265/0.335 
 No. of atoms, protein/carbohydrates/water 7 223/70/4 7 215/70/170 7 197/98/8 
 Molecules per asymmetric unit 
 RMSD from ideal    
  Bond lengths, Å 0.009 0.004 0.003 
  Bond angles, ° 1.052 0.650 0.625 
 Ramachandran plot, favored/allowed/outliers, % 87.57/10.81/1.62 92.64/7.14/0.22 87.87/9.75/2.38 
 PDB code 6CKB 6BXB 6BXF 

PDB, Protein Data Bank; RMSD, root mean square deviation.

*

The highest-resolution shell is shown in parentheses.

Rmerge = ∑hi|Ii − 〈I〉|/∑hiIi, where Ii is the observed intensity of the i-th measurement of reflection h, and 〈I〉 is the average intensity of that reflection obtained from multiple observations.

R = ∑||Fo| − |Fc||/∑|Fo|, where Fo and Fc are the observed and calculated structural factors, respectively, calculated for all data. Rfree is the R value obtained for a test set of reflections consisting of a randomly selected ∼5% subset of data excluded from refinement.

All the domains in the cβ3-E2 structure are well resolved in both molecules of the asymmetric unit (Figure 1B). As seen in the isolated αI structures, either Mg2+ or Ca2+ occupies the metal-ion–dependent adhesion site (Figure 1B). The remaining residues of the C-terminal tag after 3C protease cleavage form an antiparallel β-sheet interaction with the I-EGF2 domain (Figure 1B). Structural superimposition shows that the 2 molecules share similar conformations at the αI, hybrid, and PSI domains but have remarkably distinct conformations at the junction of I-EGF1 and I-EGF2 domains (Figure 1C). One molecule adopts an extended confirmation, whereas the other shows a bent-like conformation (Figure 1C). When compared with the native β3 headpiece structures, cβ3-E2 resembles the closed rather than open headpiece conformation (Figure 1D). However, when superimposed onto the αI domain of the cβ3 ectodomain crystal structure (A.M.M.T., Z.W., D.Z., Y.Z., Brian R. Curtis, and J.Z., manuscript submitted April 2018), a slight swing-out of the hybrid domain was observed (supplemental Figure 3A-B). This was associated with the downward displacement of the α7 helix C-terminus (supplemental Figure 3A-B). Nonetheless, the αI domain of cβ3-E2 was in a low-affinity conformation, as seen in the cβ3 ectodomain crystal structure (supplemental Figure 3A-B).

These structural features were observed in both cβ3-E2-Leu33 and cβ3-E2-Pro33 crystals. No significant structural differences were observed between these 2 isoforms of cβ3-E2 (supplemental Figure 4A-D). Therefore, the Pro33 form was used to represent the cβ3-E2 structure, because it gave a higher resolution than the Leu33 form (Table 1). Of note, the 2 cβ3-E2 molecules in the asymmetric unit packed closer to each other after crystal dehydration (supplemental Figure 2B-C), but no significant conformational changes were observed after dehydration (supplemental Figure 2B-C).

β3 extension at the I-EGF1/I-EGF2 junction

As expected, we observed an extended conformation at the I-EGF1/I-EGF2 junction in the cβ3-E2 structure. In the crystal lattice, the I-EGF2 domain of 1 molecule of the asymmetric unit was free of crystal contact and thus had enough room to adopt an extended conformation (supplemental Figure 2A). Remarkably, the I-EGF2 domain of the other molecule of the asymmetric unit was trapped in the bent-like conformation by the close lattice contacts with its neighboring symmetry mates (supplemental Figure 2A). When superimposed with the bent native β3 structure based on either the I-EGF1 or I-EGF2 domain (Figure 2A), the bent-like conformer observed in the cβ3-E2 structure actually exhibits an intermediate state in between the fully bent and fully extended conformations (Figure 2A). Therefore, we captured a snapshot of β3 integrin on the pathway from the bent to extended conformational transition.

Figure 2.

β3integrin extension at the I-EGF1/I-EGF2 junction. (A) Structural superposition of the cβ3-E2 I-EGF1-2 domains on the I-EGF1 (upper) or the I-EGF2 (lower) domain of the bent native β3 structure (PDB 4G1M). (B-D) The 3 structures are shown separately after being superimposed on the I-EGF2 domains. The bent (B), intermediate (C), and extended (D) forms are shown in cyan, green, and red, respectively. Disulfide bonds are sticks. Residues of interest are shown as Cα spheres. The 8 cysteines of the I-EGF2 domain are numbered. (E) Structural superposition on the I-EGF2 of the bent (PDB code 4NEH), intermediate (PDB 2V26), and extended (PDB 2V28) I-EGF1-2 domains of β2 integrin. (F) Structural superposition on I-EGF2 of the extended I-EGF1-2 domains of β3 (red) and β2 (green) integrins. (G-H) Structural comparison of the extended β3 I-EGF1-2 (PDB 6BXB) with the β3 I-EGF2-3 (G; PDB 3FCS) or I-EGF3-4 (H; PDB 3FCS) domains. The I-EGF3 or I-EGF4 domain was superimposed onto the I-EGF2 domain.

Figure 2.

β3integrin extension at the I-EGF1/I-EGF2 junction. (A) Structural superposition of the cβ3-E2 I-EGF1-2 domains on the I-EGF1 (upper) or the I-EGF2 (lower) domain of the bent native β3 structure (PDB 4G1M). (B-D) The 3 structures are shown separately after being superimposed on the I-EGF2 domains. The bent (B), intermediate (C), and extended (D) forms are shown in cyan, green, and red, respectively. Disulfide bonds are sticks. Residues of interest are shown as Cα spheres. The 8 cysteines of the I-EGF2 domain are numbered. (E) Structural superposition on the I-EGF2 of the bent (PDB code 4NEH), intermediate (PDB 2V26), and extended (PDB 2V28) I-EGF1-2 domains of β2 integrin. (F) Structural superposition on I-EGF2 of the extended I-EGF1-2 domains of β3 (red) and β2 (green) integrins. (G-H) Structural comparison of the extended β3 I-EGF1-2 (PDB 6BXB) with the β3 I-EGF2-3 (G; PDB 3FCS) or I-EGF3-4 (H; PDB 3FCS) domains. The I-EGF3 or I-EGF4 domain was superimposed onto the I-EGF2 domain.

Close modal

Each of the integrin I-EGF domains contains 8 cysteines that form disulfide bonds in the pattern of C1-C5, C2-C4, C3-C6, and C7-C8, except the I-EGF1 domain5  (Figure 2B). The C1-C5 disulfide bond and C1-C2 (C473-C486 of β3) loop of the I-EGF2 domain are pivotal in the transition from the bent to extended conformation at the I-EGF1/2 junction (Figure 2B-D). The C1-C2 loop of I-EGF2 was incompletely defined in the bent αIIbβ3 and 1 of the bent αVβ3 structures as a result of the lack of complete electron density, but a complete backbone conformation was relatively well defined in another bent αVβ3 structure (supplemental Figure 5A-C).4,5,35  In our intermediate cβ3-E2 structure, the strong electron density allowed us to build a complete conformation for both the backbone and side chains of the C1-C2 loop in the I-EGF2 domain (supplemental Figure 5D). We were also able to build a complete backbone conformation of the C1-C2 loop in the extended I-EGF2 domain, although some of the side chain conformations were not well defined because of the weak electron density (supplemental Figure 5E). Structural superimposition on the I-EGF2 domain revealed the extension of I-EGF1 afforded by the conformational rearrangement of the flexible C1-C2 loop and rotational movement at the C1 position (Figure 2B-D).

In the bent conformation, the Cα-Cα distance between the N-terminal C435 of I-EGF1 and the C-terminal E522 of I-EGF2 was 25.3 Å (Figure 2B). This distance changed to 33.6 Å in the intermediate conformation (Figure 2C) and to 45 Å in the extended conformation (Figure 2D). In coordination with these changes, the Cα-Cα distance between the C1 and C2 residues changed from 16.9 Å in the bent, to 14.9 Å in the intermediate, and to 13.8 Å in the extended states (Figure 2B-D). Along with the extension of the I-EGF1/2 junction, there was a rotational movement of the I-EGF1 domain using the C1-C5 disulfide bond as a hinge. There were large conformational rearrangements for both backbone and side chains of the I-EGF2 C1-C2 loop during the I-EGF1 extension (Figure 2B-D; supplemental Figure 4). The rotation of the C1-C5 disulfide bond of I-EGF2 accompanied with the swinging of the C1-C2 loop (Figure 2B-D). Interestingly, a small portion of the C1-C2 loop at the C2 end became α helical in the extended conformation (Figure 2D). No disulfide bond exchange was observed. All the disulfide bonds of I-EGF1 and I-EGF2 domains remained the same as in the bent conformation during the I-EGF1 extension (Figure 2B-D).

Two crystal structures of β2 fragments were determined previously.36  One contains the PSI, hybrid, and I-EGF1-2 domains and is bent at the I-EGF1/2 junction. The other contains an extra I-EGF3 domain and exhibits an extended I-EGF1/2 junction. When superimposed onto the I-EGF2 domain of the β2 ectodomain structure in the bent conformation,37  the bent β2 fragment, in fact, showed an intermediate conformation, in which the I-EGF1 orientated close to the extended state (Figure 2E). Compared with our extended β3 headpiece, the I-EGF1 in the extended β2 fragment was in a less straight position relative to the I-EGF2 domain (Figure 2F). The Cα-Cα distance between I-EGF2 C1 (C461) and C2 (C475) was 15.2 Å, which was longer than that of the extended β3 (Figure 2D). An α-helical conformation at the C2 end of the β2 I-EGF2 C1-C2 loop presented at both the bent and intermediate states, but it became less α helical in the extended state (Figure 2E-F).

Extended structure of β3 integrin

Previous crystallographic studies have determined the crystal structures of β3 ectodomain in the bent conformation and β3 headpiece (containing βI, hybrid, PSI, and I-EGF1 domains) either in the closed or open conformation.4-6,38  Of note, EM studies have suggested that the integrin headpiece extends away from the leg domains on the cell surface, either remaining in the closed conformation, the same as in the bent state, or becoming an open configuration.7,8,11  However, the conformations of I-EGF2-4 and β-tail domains likely remain unchanged after headpiece extension and leg separation, as shown by the EM structures. Our current cβ3-E2 crystal structures revealed the extension of β3 at the I-EGF1/I-EGF2 junction. Using our intermediate and extended I-EGF1/I-EGF2 structures as templates, and by superimposing the β3 headpiece structure onto the I-EGF1 domain and superimposing the I-EGF2-4 and β-tail structures onto the I-EGF2 domain, we were able to build a complete high-resolution structure of β3 ectodomain at both the intermediate and extended conformations (Figure 3A-D).

Figure 3.

Extended structure of β3integrin. (A) Two different views of the intermediate (green) and extended (red) cβ3-E2 structures superimposed on the I-EGF2 domain of the bent (cyan) native β3 structure (PDB 4G1M). (B) Intermediated (half-extended) conformation of αIIbβ3. The structure was built based on the intermediated conformation of I-EGF1-2 and shown in 2 views. The negative staining EM images shown are full-length αIIbβ3 (i),11  ectodomain of αVβ3 (ii),7  ectodomain of αIIbβ3 (iii),5  and full-length αIIbβ3 (iv).11  The images were not shown in the same scale. (C-D) Modeled extended β3 structure with closed (C) or open (D) headpiece. The model was generated by the superimposition of the extended cβ3-E2 structure onto the I-EGF2 domain of the native β3 structure first and then superimposition of the closed or open β3 headpiece structure onto the hybrid domain of the cβ3-E2 structure. (E-F) Overlay of the extended β3 structure with the EM image of extended αVβ37  with closed headpiece (E) or extended αIIbβ3 with open headpiece11  (F). (G) αIIb thigh/calf-1 interface at the bent conformation (PDB 3FCS). Interfacial residues are shown as sticks with green and cyan carbons for thigh and calf-1 domains, respectively. Hydrogen bonds are dashed in magenta. Disulfide bonds are yellow sticks. Panels Bi, Biv, and F reprinted from Eng et al11  with permission; panels Bii and E are reprinted from Takagi et al7  with permission; and panel Biii is reprinted from Zhu et al5  with permission.

Figure 3.

Extended structure of β3integrin. (A) Two different views of the intermediate (green) and extended (red) cβ3-E2 structures superimposed on the I-EGF2 domain of the bent (cyan) native β3 structure (PDB 4G1M). (B) Intermediated (half-extended) conformation of αIIbβ3. The structure was built based on the intermediated conformation of I-EGF1-2 and shown in 2 views. The negative staining EM images shown are full-length αIIbβ3 (i),11  ectodomain of αVβ3 (ii),7  ectodomain of αIIbβ3 (iii),5  and full-length αIIbβ3 (iv).11  The images were not shown in the same scale. (C-D) Modeled extended β3 structure with closed (C) or open (D) headpiece. The model was generated by the superimposition of the extended cβ3-E2 structure onto the I-EGF2 domain of the native β3 structure first and then superimposition of the closed or open β3 headpiece structure onto the hybrid domain of the cβ3-E2 structure. (E-F) Overlay of the extended β3 structure with the EM image of extended αVβ37  with closed headpiece (E) or extended αIIbβ3 with open headpiece11  (F). (G) αIIb thigh/calf-1 interface at the bent conformation (PDB 3FCS). Interfacial residues are shown as sticks with green and cyan carbons for thigh and calf-1 domains, respectively. Hydrogen bonds are dashed in magenta. Disulfide bonds are yellow sticks. Panels Bi, Biv, and F reprinted from Eng et al11  with permission; panels Bii and E are reprinted from Takagi et al7  with permission; and panel Biii is reprinted from Zhu et al5  with permission.

Close modal

At the intermediate state, the β3 headpiece extended halfway to the fully extended conformation (Figure 3A). In addition to the acute bent conformation (Figure 3Bi), the intermediate conformations of αVβ3 (Figure 3Bii) and αIIbβ3 complexes (Figure 3Biii-iv) were observed by the EM studies.5,7,11  At this intermediate state, the leg domains were still associated with each other, as in the bent conformation, and the headpiece remained closed. The structural model of the αIIb subunit at the intermediate conformation was built by the structural superimpositions of the closed αIIbβ3 headpiece and the associated αIIbβ3 leg domains onto the modeled intermediated β3 subunit with the closed headpiece (Figure 3B). Remarkably, this structural model of αIIbβ3 in the intermediate conformation agreed well with the intermediate EM structures (Figure 3B), demonstrating the reliability of our structure.

On the basis of the extended cβ3-E2 structure, we built the extended structure of the β3 ectodomain with either the closed (Figure 3C) or the open (Figure 3D) headpiece conformation. Similar to the intermediate conformation of β3, the extended structures of β3 fit near perfectly into the EM structures of extended β3 with a closed or open headpiece (Figure 3E-F), again demonstrating the reliability of our structural models. We did not model the extended conformation of αIIb ectodomain because of the lack of αIIb and αV crystal structures at the extended state.

Structure of the β3 PSI domain at the bent and extended conformations

The β3 PSI domain has been shown to have some functional relevance.39,40  Of interest, a Leu33Pro polymorphism at the β3 PSI domain forms the HPA-1a/b alloantigen.15,17  The β3 PSI domain also contains the epitope of active conformation-specific mAb AP5,41  indicating its conformational changes upon β3 activation. Leu33Pro locates at the C26-C38 loop of the PSI domain (Figure 4A-B). Previous studies have suggested that β3 harboring Pro33 could be a risk factor for thrombosis because of the elevated function of αIIbβ3 in platelets, but the conclusions are controversial.18-20  Our structures of the β3 headpiece in both Leu33 and Pro33 forms as well as in the intermediate and extended states provide new structural information of the PSI domain.

Figure 4.

Structure of the β3PSI domain. (A) Two different views of the superposition of the bent (PDB 3FCS), intermediate, and extended β3 structures on the PSI domain. Disulfide bonds, Pro33, and Leu33 are sticks. Selected cysteines are shown as Cα spheres. (B) Sequence alignment of a portion of the PSI domain containing the C26-C38 loop. Disulfide linkages are indicated. (C) Mn2+-stimulated binding of the ligand mimetic mAb PAC-1 to HEK293FT cells transfected with the indicated αIIb and β3 constructs. PAC-1 binding was performed in the presence of 1 mM of Ca2+/Mg2+ or 0.2 mM of Ca2+ plus 2 mM Mn2+. (D) Talin head (TH)–induced PAC-1 binding to HEK293FT cells transfected with the indicated αIIb and β3 constructs plus EGFP or EGFP-TH. PAC-1 binding was performed in the presence of 1 mM of Ca2+/Mg2+. (E) Mn2+-stimulated binding of fibronectin (Fn) to HEK293FT cells transfected with the indicated αV and β3 constructs. Fn binding was performed in the presence of 1 mM of Ca2+/Mg2+ or 0.2 mM of Ca2+ plus 2 mM of Mn2+. All the ligand binding results are presented as the mean fluorescence intensity (MFI) normalized to integrin expression. WT, wild type.

Figure 4.

Structure of the β3PSI domain. (A) Two different views of the superposition of the bent (PDB 3FCS), intermediate, and extended β3 structures on the PSI domain. Disulfide bonds, Pro33, and Leu33 are sticks. Selected cysteines are shown as Cα spheres. (B) Sequence alignment of a portion of the PSI domain containing the C26-C38 loop. Disulfide linkages are indicated. (C) Mn2+-stimulated binding of the ligand mimetic mAb PAC-1 to HEK293FT cells transfected with the indicated αIIb and β3 constructs. PAC-1 binding was performed in the presence of 1 mM of Ca2+/Mg2+ or 0.2 mM of Ca2+ plus 2 mM Mn2+. (D) Talin head (TH)–induced PAC-1 binding to HEK293FT cells transfected with the indicated αIIb and β3 constructs plus EGFP or EGFP-TH. PAC-1 binding was performed in the presence of 1 mM of Ca2+/Mg2+. (E) Mn2+-stimulated binding of fibronectin (Fn) to HEK293FT cells transfected with the indicated αV and β3 constructs. Fn binding was performed in the presence of 1 mM of Ca2+/Mg2+ or 0.2 mM of Ca2+ plus 2 mM of Mn2+. All the ligand binding results are presented as the mean fluorescence intensity (MFI) normalized to integrin expression. WT, wild type.

Close modal

Structural superimposition on the I-EGF1 domain showed that the overall conformations of the PSI and I-EGF1 domains were nearly identical between the intermediate and bent states of β3 (Figure 4A). Interestingly, in the extended conformation, the C26-C38 loop of the PSI domain moved close to the I-EGF1 domain (Figure 4A). In addition, the C437-C448 loop of I-EGF1 domain also moved toward the PSI C26-C38 loop in the extended conformation (Figure 4A). These structural characters were observed in the cβ3-E2 structures with either Pro33 or Leu33 (supplemental Figure 4C-D), demonstrating that Leu33Pro substitution does not affect the local conformation of PSI domain.

Leu33Pro substitution does not elevate the ligand binding of β3 integrin

The sequences of the C26-C38 loop are relatively conserved among different species, but variations are seen at the positions equivalent to β3-Leu33 (Figure 4B). We examined whether the Leu33Pro polymorphism affects the activation of β3 integrin. Ligand mimetic mAb PAC-1 was used to measure the activation of αIIbβ3 stimulated either by Mn2+ or by the cytoplasmic activating mutation αIIb-R995A that mimics inside-out activation.42  No differences in PAC-1 binding were observed between β3-Leu33 and β3-Pro33 (Figure 4C). Similarly, no difference was observed in PAC-1 binding to αIIbβ3-Leu33 and αIIbβ3-Pro33 induced by the overexpression of EGFP-tagged talin head domain (Figure 4D). Moreover, no difference was found between β3-Leu33 and β3-Pro33 in Mn2+-stimulated fibronectin binding to αVβ3 (Figure 4E). These data demonstrate that Pro33 does not render β3 integrin more active than the Leu33 form.

A conserved arginine at the β subunit PSI/hybrid domain interface restrains integrin activation

The β3 PSI domain connects with the hybrid domain through a short loop (Figure 5A). During the headpiece extension and hybrid domain swing-out, the PSI domain moves along with the hybrid domain (supplemental Figure 1C). We found no significant structural differences at the PSI/hybrid interface between the bent and extended β3 headpieces (Figure 5A-B). The side chain of Arg93 at the hybrid domain A’B loop inserts into the hybrid/PSI interface and forms multiple hydrogen bonds with the backbone carbonyl oxygens of the PSI domain and the hybrid-PSI connecting loop (Figure 5A). These hydrogen bonds are maintained in the extended β3 conformation (Figure 5B) and in the open headpiece of β3.6  Such hydrogen bond interactions may be critical in maintaining the rigidity of the hybrid/PSI interface. Of interest, the equivalent Arg is completely conserved among all the integrin β subunits (Figure 5C). Mutagenesis studies were performed to investigate the function of this conserved Arg in integrin activation. As shown by the ligand-binding assay, β3-R93A and β3-R93Q mutations remarkably augmented Mn2+-stimulated PAC-1 binding to αIIbβ3 integrin, although the expression level was also greatly decreased (Figure 5D). Basal PAC-1 binding under the physiological Ca2+/Mg2+ concentration was also increased by β3-R93 mutations (Figure 5D). Consistently, the β3-R93A mutation greatly enhanced fibronectin binding to αVβ3 (Figure 5E). Similar results were obtained for β2 and β1 integrins. The β2-R87A mutation greatly enhanced ICAM-1 binding to αLβ2 (Figure 5F). β1-R104A greatly enhanced fibronectin binding to α5β1 (Figure 5G). Consistent with the fibronectin-binding assay, the β1-R104A mutation enhanced both basal- and Mn2+-stimulated binding of 9EG7 to α5β1 (Figure 5H), demonstrating that disruption of the hybrid/PSI interface induces the extension of β1 integrin.

Figure 5.

A conserved arginine at the hybrid-PSI interface. (A-B) The hybrid-PSI interface of bent (A) and extended (B) β3. Disulfide bonds are yellow sticks. Hydrogen bonds are dashed in magenta. (C) Sequence alignment of the hybrid domain A’B loop. (D-H) PAC-1, ICAM-1, fibronectin (Fn), or 9EG7 binding of the β3-R93 (D-E), β2-R87 (F), or β1-R104 (G-H) mutations coexpressed with αIIb (D), αV (E), αL (F), or α5 (G-H) integrin in HEK293FT cells. The binding was performed in the presence of 1 mM of Ca2+/Mg2+ (Ca/Mg) or 0.2 mM of Ca2+ plus 2 mM Mn2+ (Ca/Mn) and is presented as mean fluorescence intensity (MFI) normalized to integrin expression. One representative result is shown. WT, wild type.

Figure 5.

A conserved arginine at the hybrid-PSI interface. (A-B) The hybrid-PSI interface of bent (A) and extended (B) β3. Disulfide bonds are yellow sticks. Hydrogen bonds are dashed in magenta. (C) Sequence alignment of the hybrid domain A’B loop. (D-H) PAC-1, ICAM-1, fibronectin (Fn), or 9EG7 binding of the β3-R93 (D-E), β2-R87 (F), or β1-R104 (G-H) mutations coexpressed with αIIb (D), αV (E), αL (F), or α5 (G-H) integrin in HEK293FT cells. The binding was performed in the presence of 1 mM of Ca2+/Mg2+ (Ca/Mg) or 0.2 mM of Ca2+ plus 2 mM Mn2+ (Ca/Mn) and is presented as mean fluorescence intensity (MFI) normalized to integrin expression. One representative result is shown. WT, wild type.

Close modal

Our high-resolution structural studies reveal how β3 becomes extended at the I-EGF1/2 junction. Our data suggest that the multiple conformational states of β3 observed in many EM studies5,8-11,43-45  could be afforded by the conformational plasticity of the I-EGF2 C1-C2 loop. Previous studies have demonstrated that the length of the I-EGF2 C1-C2 loop modulates β3 integrin activation.46  The long and short loops favor the resting and active states, respectively.46  The C1-C2 loop of I-EGF2 was the longest among the I-EGF domains. The I-EGF3 C1-C2 loop had 6 residues, whereas the I-EGF4 C1-C2 loop had 4 residues. The angle between I-EGF3 and I-EGF4 was closer to straight than the others (Figure 2G-H). The C1-C2 loop of β2 I-EGF2 had 13 residues (Figure 2F). Consistently, the β2 I-EGF1/2 junction was less extended than that of β3, which had 12 residues spanning the C1-C2 loop (Figure 2F). These structural features of I-EGF domains are compatible with the long-range conformational changes of integrin. The rigidity of I-EGF2/3 and I-EGF3/4 interfaces provides a support for the extension of the integrin headpiece and for the resistance of mechanical force.5  The flexibility of the I-EGF1/2 interface allows the headpiece to pose at different conformations, which is required for affinity regulation. Given the critical role of the I-EGF2 C1-C2 loop in integrin extension, an intriguing strategy would be to target the C1-C2 loop, such as by antibodies, which may modulate integrin conformational change and cellular function.

Our structural studies have important implications for integrin conformational activation. Integrin headpiece extension is afforded by the extension of the β subunit at the I-EGF1/2 junction (β knee) and the extension of the α subunit at the thigh/calf-1 junction (α knee; Figure 3B-F; supplemental Figure 1). Because of the large interface formed between the β-propeller and βI domains, the α and β extensions need to occur simultaneously. Our high-resolution structures of β3- knee at intermediate and extended conformations demonstrate the flexibility of this region. Extension of the I-EGF1 domain did not generate new interactions at the I-EGF1/2 junction (Figure 3D). Indeed, only a very small interface formed at the β3 I-EGF1/2 junction, even in the bent conformation (Figure 3B). By contrast, the thigh/calf-1 junction in bent αIIb formed a larger interface composed of hydrogen bonds and hydrophobic interactions (Figure 3G). This suggests that the bent αIIb knee may resist the force of extension more than β3 knee. EM and our modeled structure show that the αIIb and αV subunits can adopt an intermediate (half-extended) conformation (Figure 3B). After complete extension, α knee becomes straight and the thigh and calf-1 domains are expected to form new close contacts at their junction. Unlike extended β3 knee, the new interface of extended α knee is expected to be rigid. These structural features are important for the conformational regulation of integrin by cytoskeletal force under conditions of cell adhesion and migration.5,47,48  The flexibility of an extended β subunit afforded by the flexible I-EGF1/2 junction is required to sense and resist the changes of cellular forces, whereas the rigidity of an extended α leg may provide support for the sensing and resisting of tensile force by the β subunit.47,49-51 

Local conformational changes were observed at the PSI and I-EGF1 domains upon β3 extension. It is not readily known whether such conformational changes are functionally significant. Nonetheless, structural diversities of the PSI and I-EGF1 domains, especially at the C26-C38 loop, have been observed among the reported β3 crystal structures (A.M.M.T., Z.W., D.Z., Y.Z., Brian R. Curtis, and J.Z., manuscript submitted April 2018). Moreover, the biological relevance of such conformational diversities is implicated by the binding of heterogeneous anti–HPA-1a alloantibodies. All anti–HPA-1a alloantibodies require Leu33 at the β3 PSI C26-C38 loop.16,17  However, some anti–HPA-1a alloantibodies require noncontiguous sequences and the integrity of the PSI domain to form the HPA-1a epitope.52-56  In particular, certain anti–HPA-1a alloantibodies require both the PSI and I-EGF1 domains.52,56,57  We have unpublished data demonstrating that the specific residues of the I-EGF1 domain are required for the binding of certain anti–HPA-1 alloantibodies. Of significance, recent studies have demonstrated that a subtype of maternal anti–HPA-1a alloantibodies could distinguish the αIIbβ3 vs αVβ3 complex.58  αVβ3-specific anti–HPA-1a alloantibodies were indicated to be the cause of intracranial hemorrhages in fetus and neonates through the blockage of the αVβ3 function in endothelial cells.58  We have data showing that αIIbβ3-specific anti–HPA-1a alloantibodies can specifically recognize the bent but not extended conformation of β3 (A.M.M.T., Z.W., D.Z., Y.Z., Brian R. Curtis, and J.Z., manuscript submitted April 2018). In addition, a recent study showed that EDTA treatment of αIIbβ3, known to disturb αIIbβ3 conformation,41,59-61  reduced the binding of anti–HPA-1a.62  All these data suggest the conformation-dependent feature of anti–HPA-1a alloantibodies. The conformational changes of the PSI and I-EGF1 domains observed in our extended β3 structure may account for the differences in anti–HPA-1a binding.

It is remarkable that the anti–HPA-1a alloantibodies could distinguish the subtle conformational differences of the PSI and I-EGF1 domains. More interestingly, some but not all anti–HPA-1a alloantibodies were found to block the function of αIIbβ3 and/or αVβ3 integrins.58,63  Our current studies provide a structure-based interpretation for this observation. The conformational differences of the PSI and I-EGF1 domains may determine the binding poses of the alloantibodies, which in turn exert either a blocking or nonblocking effect on β3 function. In particular, the bent conformation-dependent anti–HPA-1a alloantibodies may block the integrin function by stabilizing β3 in the bent conformation. This raises an intriguing possibility that the inhibitory antibodies targeting the PSI and I-EGF1 domains may be developed to specifically target αIIbβ3 or αVβ3 integrin.

Polymorphism substitution β3-R93Q has been shown to disrupt the binding of certain anti–HPA-1a (β3-Leu33) alloantibodies.64  β3-R93 is more than 28-Å distal from β3-Leu33 and is buried into the hybrid/PSI interface, and thus cannot directly participate in the HPA-1a epitope. Here, we found that β3-R93 mutations induced the active conformation of β3 integrin, which may have led to the structural changes of the PSI C26-C38 loop and the I-EGF1 domain as observed in our extended β3 structure (Figure 4A). As discussed, such conformational changes may alter the binding of anti–HPA-1a alloantibodies.

We found no differences in either conformation or ligand binding between the Leu33 and Pro33 forms of β3. Studies using human platelets or cell lines have shown that β3-Pro33 enhanced cell adhesion and outside-in signaling.18,65-68  A recent study using knock-in mice showed the Pro33 form of mouse β3 increased platelet adhesion, clot formation, and aggregation, probably as a result of elevated Src activation.69  However, agonist-induced soluble ligand binding was not affected, consistent with our data. Interestingly, the talin association with mouse β3 was also increased by the presence of Pro33.69  Because Pro33 does not directly alter the conformation of the PSI domain (Figure 4A), it remains unknown by which mechanism Pro33 affects β3 function. Molecular dynamics simulation studies have indicated that Pro33 substitution might affect β3 function by modifying the structural equilibrium of the PSI and the I-EGF domains and probably integrin extension,70,71  but such an effect may not directly affect ligand binding according to our study.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

The authors thank Peter Newman for helpful discussions and for providing antibodies.

This work was supported by grants HL122985 and HL131836 (J.Z.) from the National Heart, Lung, and Blood Institute of the National Institutes of Health.

Contribution: D.Z. collected X-ray diffraction data and refined the structures; A.M.M.T. performed functional assays and collected X-ray diffraction data; Y.Z. contributed to protein purification; Z.W. contributed to the generation of DNA constructs; J.Z. designed the study, did protein crystallography, and wrote the manuscript; and all authors contributed to manuscript preparation.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Jieqing Zhu, Blood Research Institute, BloodCenter of Wisconsin, 8727 Watertown Plank Rd, Milwaukee, WI 53226; e-mail: jieqing.zhu@bcw.edu.

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