Open Headpiece Research Articles

Ligand binding initiates single-molecule integrin conformational activation

Integrins link the extracellular environment to the actin cytoskeleton in cell migration and adhesiveness. Rapid coordination between events outside and inside the cell is essential. Single-molecule fluorescence dynamics show that ligand binding to the bent-closed integrin conformation, which predominates on cell surfaces, is followed within milliseconds by two concerted changes, leg extension and headpiece opening, to give the high-affinity integrin conformation. The extended-closed integrin conformation is not an intermediate but can be directly accessed from the extended-open conformation and provides a pathway for ligand dissociation. In contrast to ligand, talin, which links the integrin β-subunit cytoplasmic domain to the actin cytoskeleton, modestly stabilizes but does not induce extension or opening. Integrin activation is thus initiated by outside-in signaling and followed by inside-out signaling. Our results further imply that talin binding is insufficient for inside-out integrin activation and that tensile force transmission through the ligand-integrin-talin-actin cytoskeleton complex is required.

Magnesium enhances the graft-versus-tumor effect of donor lymphocytic infusion on hematologic malignancies.

Donor lymphocyte infusion (DLI) cures relapsed hematologic malignancies after allogeneic hematopoietic stem cell transplantation through the graft-versus-tumor (GVT) effect. Although the important role of magnesium in enhancing immunity has been mentioned in studies, limited clinical data have explored how magnesium affects the efficacy of DLI. Besides, although laboratory data demonstrate that magnesium can enhance CD8+ T cells effector function, whether magnesium regulates the tumor killing effect of peripheral blood mononuclear cells (PBMCs) remains to be explored. Here, for the retrospective study, we collected clinical data of relapsed patients receiving DLI and explored the relationship between different serum magnesium levels and patient outcomes. For in vitro studies, we investigated the effect of magnesium on the cytotoxicity of DLI cells which were PBMCs and preliminarily explored the mechanism. Eighty-one patients were enrolled in this study. It was found that the high post-DLI magnesium level was significantly associated with a higher incidence of complete remission (CR) or partial remission (CR/PR) and a higher possibility of survival. The magnesium level after DLI was an independent risk factor of overall survival. In vitro studies proved that increased magnesium enhanced the cytotoxic function of PBMCs on hematologic malignancies. Besides, magnesium modulated LFA-1 headpiece opening. When blocking the integrin-ligand interaction between LFA-1 and ICAM-1, the regulation effect of magnesium on PBMCs was weakened. Therefore, it was possible that magnesium regulated PBMCs effector function by stimulating LFA-1. These results show that serum magnesium levels affect immunological responses mediated by donor lymphocytes in hematologic malignancies.

Structure of an Extended β Integrin

Integrins are a family of 24 α/β heterodimeric adhesion receptors that transmit bidirectional signals across the cell membrane. Each integrin subunit contains a large extracellular domain composed of headpiece and leg piece, a transmembrane domain and usually a short cytoplasmic tail. The α headpiece consists of the common β-propeller and thigh domains, and an extra αI domain inserted into the β-propeller domain in a subset of α subunits such as αL. The α leg piece includes calf-1 and -2 domains. The β headpiece contains βI, which is a structural homolog of αI domain, hybrid, PSI, and I-EGF1 domains. The β leg piece contains I-EGF2-4 and β-tail domains. Structural studies in the last 15 years, largely based on the two β3 integrins, αIIbβ3 and αVβ3, greatly advanced our understanding of integrin conformational regulation upon activation. The platelet specific αIIbβ3 plays an essential role in hemostasis and thrombosis, while the αVβ3 is important for the growth and survival of many cell types. In the resting state, integrin adopts a bent conformation with the headpiece folded onto the leg piece. Crystallographic and EM studies of αIIbβ3 and αVβ3 revealed two 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. Despite the visualization of β3 extension by EM at very low resolution, a high-resolution extended β3 structure has been missing.A Leu33Pro polymorphism at the β3 PSI domain defines the human platelet alloantigen 1 (HPA-1) system. The mismatched HPA-1a/b (Leu33/Pro33) is a major cause of two severe bleeding disorders, fetal/neonatal alloimmune thrombocytopenia (FNAIT) and post-transfusion purpura (PTP) due to the generation of anti-HPA-1 alloantibodies. In addition, it has been controversial whether the Pro33 variant can enhance β3 activity, being a risk factor for thrombosis. It is also unknown whether the Leu33Pro substitution affects the conformation of PSI domain, which undergoes outward movement along with the headpiece opening and extension.It has been difficult to obtain the crystal structure of a full-length extended integrin largely due to the conformational heterogeneity and flexibility, especially in the lower leg domains. In this study, we engineered a chimeric β3 fragment, denoted as cβ3-E2, including the domains of PSI, hybrid, I-EGF1, I-EGF2, and the βI replaced with the αL αI domain, which enables the expression of β3 in the absence of α subunit. We determined the crystal structures of cβ3-E2 in both Leu33 and Pro33 forms. Strikingly, two conformations of cβ3-E2, an intermediate state and a fully extended state are simultaneously present in one crystal. The β3 extension occurs at the I-EGF1 and I-EGF2 interface, denoted as β-knee. Our structure revealed the structural changes of the flexible loop, C473-C486 or C1-C2 loop of I-EGF2 in the β-knee, which has been shown to modulate integrin activation by mutagenesis studies. No significant structural differences were observed between the PSI domains having Leu33 and Pro33. We next compared the ligand binding of β3-Leu33 and β3-Pro33 induced by Mn2+ or talin head domain. No significant differences were observed either in αIIbβ3 or αVβ3, indicating that the Leu33Pro substitution may not affect β3 integrin activation. However, the PSI C26-C38 loop harboring the Leu33Pro polymorphism moves closer to the I-EGF1 domain in the extended conformation. In addition, we found that a completely conserved arginine (Arg93 in β3) at the PSI/hybrid domain interface is important for maintaining the resting integrin state since the alanine or glutamine substitution renders β1, β2 and β3 integrin hyperactive. Interestingly, a naturally occurring β3-Arg93Gln substitution has been shown to disrupt the HPA-1a epitope. Finally, based on our cβ3-E2 structure, we built the extended full-length β3 structures with either closed or open headpiece, which can perfectly fit into the EM structures of extended αIIbβ3 with either closed or open headpiece, respectively. In summary, our study provides structural details of an extended β3 integrin, showing the conformational changes at the PSI, I-EGF1 and I-EGF2 domains, demonstrating their importance in regulating integrin activation and alloimmune response. DisclosuresNo relevant conflicts of interest to declare.

Effects of the association of the αv β8 lower legs on integrin ligand binding.

Many integrins transmit signals through global conformational changes. However, it is unclear whether integrin αv β8 adopts a similar mechanism during integrin activation and signaling on the cell surface. Here, we showed that disulfide-bonded mutants, which prevented integrin αv β8 lower leg dissociation, bound ligands with similar level as the wild-type protein, suggesting that αv β8 ligand binding did not require lower leg disassociation. We further showed that the N-glycosylation mutant at the interface between the β I and hybrid domains did not affect ligand binding, suggesting that the αv β8 open headpiece was not present on the cell surface. We proposed that αv β8 integrin may adopt only one state, that is, the extended conformation with a closed headpiece. Our results showed that two lower legs retained heterodimeric interfaces, and this association might be important for stabilizing integrin in the extended conformation. Therefore, αv β8 may not transmit bidirectional signals across the plasma membrane but instead may serve as an anchoring site with high affinity and high accessibility for extracellular ligands.

Inactivation of HeLa cells on nanoporous gold

Nanoporous metals strongly affect organic matter; however, there is a poor understanding of their effects on cells. The present work shows that HeLa cells on nanoporous gold (NPG) were less active than those on flat gold (FG) with no nanoporous structure. Initially, HeLa cells adhered to the NPG over a period of more than 10 h, then the adhered cells subsequently exhibited apoptosis that was not related to anoikis. ELISA analyses showed that the conformational change of fibronectin was more greatly induced by NPG than FG. First-principles calculations and molecular dynamics simulations were performed to investigate the conformational change in the RGD sequence and the integrin signaling. The simulations suggested that the extended form of integrin, with an open headpiece, was not generated owing to the conformational change of RGD, and the outside-in signals could not be intracellularly transmitted via the integrin binding to the fibronectin on NPG, resulting in cell death.

Ligand- and cation-induced structural alterations of the leukocyte integrin LFA-1

In αI integrins, including leukocyte function-associated antigen 1 (LFA-1), ligand-binding function is delegated to the αI domain, requiring extra steps in the relay of signals that activate ligand binding and coordinate it with cytoplasmic signals. Crystal structures reveal great variation in orientation between the αI domain and the remainder of the integrin head. Here, we investigated the mechanisms involved in signal relay to the αI domain, including whether binding of the ligand intercellular adhesion molecule-1 (ICAM-1) to the αI domain is linked to headpiece opening and engenders a preferred αI domain orientation. Using small-angle X-ray scattering and negative-stain EM, we define structures of ICAM-1, LFA-1, and their complex, and the effect of activation by Mn2+ Headpiece opening was substantially stabilized by substitution of Mg2+ with Mn2+ and became complete upon ICAM-1 addition. These agents stabilized αI-headpiece orientation, resulting in a well-defined orientation of ICAM-1 such that its tandem Ig-like domains pointed in the opposite direction from the β-subunit leg of LFA-1. Mutations in the integrin βI domain α1/α1' helix stabilizing either the open or the closed βI-domain conformation indicated that α1/α1' helix movements are linked to ICAM-1 binding by the αI domain and to the extended-open conformation of the ectodomain. The LFA-1-ICAM-1 orientation described here with ICAM-1 pointing anti-parallel to the LFA-1 β-subunit leg is the same orientation that would be stabilized by tensile force transmitted between the ligand and the actin cytoskeleton and is consistent with the cytoskeletal force model of integrin activation.

A pivotal role for a conserved bulky residue at the α1-helix of the αI integrin domain in ligand binding

The ligand-binding βI and αI domains of integrin are the best-studied von Willebrand factor A domains undergoing significant conformational changes for affinity regulation. In both βI and αI domains, the α1- and α7-helixes work in concert to shift the metal-ion-dependent adhesion site between the resting and active states. An absolutely conserved Gly in the middle of the α1-helix of βI helps maintain the resting βI conformation, whereas the homologous position in the αI α1-helix contains a conserved Phe. A functional role of this Phe is structurally unpredictable. Using αLβ2 integrin as a model, we found that the residue volume at the Phe position in the α1-helix is critical for αLβ2 activation because trimming the Phe by small amino acid substitutions abolished αLβ2 binding with soluble and immobilized intercellular cell adhesion molecule 1. Similar results were obtained for αMβ2 integrin. Our experimental and molecular dynamics simulation data suggested that the bulky Phe acts as a pawl that stabilizes the downward ratchet-like movement of β6-α7 loop and α7-helix, required for high-affinity ligand binding. This mechanism may apply to other von Willebrand factor A domains undergoing large conformational changes. We further demonstrated that the conformational cross-talk between αL αI and β2 βI could be uncoupled because the β2 extension and headpiece opening could occur independently of the αI activation. Reciprocally, the αI activation does not inevitably lead to the conformational changes of the β2 subunit. Such loose linkage between the αI and βI is attributed to the αI flexibility and could accommodate the αLβ2-mediated rolling adhesion of leukocytes.

Conformational equilibria and intrinsic affinities define integrin activation

We show that the three conformational states of integrin α5β1 have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000‐fold higher affinity of the extended‐open state than the bent‐closed and extended‐closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact α5β1. On the surface of K562 cells, α5β1 is 99.8% bent‐closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N‐glycans and leg domains in each subunit that connect the ligand‐binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single‐span transmembrane domains.

Relating conformation to function in integrin α5β1

Whether β1 integrin ectodomains visit conformational states similarly to β2 and β3 integrins has not been characterized. Furthermore, despite a wealth of activating and inhibitory antibodies to β1 integrins, the conformational states that these antibodies stabilize, and the relation of these conformations to function, remain incompletely characterized. Using negative-stain electron microscopy, we show that the integrin α5β1 ectodomain adopts extended-closed and extended-open conformations as well as a bent conformation. Antibodies SNAKA51, 8E3, N29, and 9EG7 bind to different domains in the α5 or β1 legs, activate, and stabilize extended ectodomain conformations. Antibodies 12G10 and HUTS-4 bind to the β1 βI domain and hybrid domains, respectively, activate, and stabilize the open headpiece conformation. Antibody TS2/16 binds a similar epitope as 12G10, activates, and appears to stabilize an open βI domain conformation without requiring extension or hybrid domain swing-out. mAb13 and SG/19 bind to the βI domain and βI-hybrid domain interface, respectively, inhibit, and stabilize the closed conformation of the headpiece. The effects of the antibodies on cell adhesion to fibronectin substrates suggest that the extended-open conformation of α5β1 is adhesive and that the extended-closed and bent-closed conformations are nonadhesive. The functional effects and binding sites of antibodies and fibronectin were consistent with their ability in binding to α5β1 on cell surfaces to cross-enhance or inhibit one another by competitive or noncompetitive (allosteric) mechanisms.

β-Subunit Binding Is Sufficient for Ligands to Open the Integrin αIIbβ3 Headpiece

The platelet integrin αIIbβ3 binds to a KQAGDV motif at the fibrinogen γ-chain C terminus and to RGD motifs present in loops in many extracellular matrix proteins. These ligands bind in a groove between the integrin α and β-subunits; the basic Lys or Arg side chain hydrogen bonds to the αIIb-subunit, and the acidic Asp side chain coordinates to a metal ion held by the β3-subunit. Ligand binding induces headpiece opening, with conformational change in the β-subunit. During this opening, RGD slides in the ligand-binding pocket toward αIIb, with movement of the βI-domain β1-α1 loop toward αIIb, enabling formation of direct, charged hydrogen bonds between the Arg side chain and αIIb. Here we test whether ligand interactions with β3 suffice for stable ligand binding and headpiece opening. We find that the AGDV tetrapeptide from KQAGDV binds to the αIIbβ3 headpiece with affinity comparable with the RGDSP peptide from fibronectin. AGDV induced complete headpiece opening in solution as shown by increase in hydrodynamic radius. Soaking of AGDV into closed αIIbβ3 headpiece crystals induced intermediate states similarly to RGDSP. AGDV has very little contact with the α-subunit. Furthermore, as measured by epitope exposure, AGDV, like the fibrinogen γ C-terminal peptide and RGD, caused integrin extension on the cell surface. Thus, pushing by the β3-subunit on Asp is sufficient for headpiece opening and ligand sliding, and no pulling by the αIIb subunit on Arg is required.

Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3.

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, p1YMESRADR8, corresponding to amino acids 313–320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (pE315-pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.

The Dual Structural Roles of the Membrane Distal Region of α Integrin Cytoplasmic Tail in Integrin inside-out Activation

Integrin inside-out activation is essential for platelet aggregation mediated by αIIbβ3 and leukocytes migration and arresting mediated by αLβ2. How integrin is activated by the inside-out stimulation is not completely understood. Integrin activation from inside the cell is regulated through the transmembrane and cytoplasmic domains. Mutagenesis and structural studies revealed that the inactive integrin conformation is maintained by the specific interactions at the transmembrane and cytoplasmic domains. Inside-out signals impinging on integrin cytoplasmic domain disturb the transmembrane and cytoplasmic associations, resulting in conformational change of extracellular domain that is required for binding ligands. Studies on the mechanism of integrin inside-out activation have been focused on β cytoplasmic tail that is relatively conserved and bears binding sites for the common intracellular activators including talin and kindlin. The integrin α cytoplasmic tails only share a conserved GFFKR motif at the membrane-proximal region that forms specific interface with the membrane-proximal region of β cytoplasmic tail. The membrane-distal regions after the GFFKR motif are diverse significantly both in length and sequence. Their roles in integrin activation have not been well characterized. In this study, by comprehensive mutagenesis, we defined the role of the membrane-distal region of α integrin cytoplasmic tail in maintaining integrin in the resting state and in integrin inside-out activation. We found that complete deletion of the αIIb cytoplasmic membrane-distal region greatly enhances αIIbβ3 activation induced by the active mutations such as β3-K716A and β3-G708L, indicating that the missing of membrane-distal region facilitates integrin activation, i.e. the αIIb membrane-distal region contributes to the inactive integrin conformation. On the other hand, complete deletion of the αIIb membrane-distal region abolished integrin activation induced by the active mutations of αIIb-R995 and β3-D723, indicating that the αIIb membrane-distal region also contributes to integrin inside-out activation. We demonstrated that deletion of the membrane-distal region of αIIb, αV, or αL integrin greatly diminished ligand binding induced by overexpression of talin-1 head and/or kindlin-2 or -3 in 293FT cells. We further confirmed the effect of α cytoplasmic membrane-distal region on integrin inside-out activation in K562 cells. In the absence of αIIb cytoplasmic membrane-distal region, PMA failed to induce ligand binding to αIIbβ3 integrin expressed in K562 cells. This effect was due to the lack of talin-1-head and kindlin-induced integrin conformational change (ectodomain extension and headpiece opening) in the absence of α cytoplasmic membrane-distal region as reported by the conformation-dependent monoclonal antibodies. Structural superposition of αIIbβ3 transmembrane-cytoplasmic heterodimer and talin-1-head/β-tail complex reveals steric clashes between talin-1 head and the αIIb membrane-distal residues (NR997) immediately follow the GFFKR motif, which has been suggested to play a role in talin-mediated integrin activation. To test this possibility, we retained two native residues, NR997 for the αIIb membrane-distal region and found that it partially restores talin-1-head-induced integrin activation. Replacing the NR997 with small amino acids, GG997 or AA997 has little effect, while with the bulky residues YY997 significantly reduced talin-1-head-induced αIIbβ3 activation. Interestingly, retaining two native residues for the membrane-distal region of αV or αL integrin failed to restore talin-1-head-induced αVβ3 or αLβ2 activation. Retaining as long as 8 native residues for the αL membrane-distal region is not sufficient to restore talin-1-head-induced αLβ2 activation to the level of intact αL. These data demonstrate that a steric clash might play a role but is not the sole mechanism by which the α cytoplasmic membrane-distal region participates in integrin inside-out activation. A proper length and amino acids of the membrane-distal region is required for talin-induced integrin activation. Our data established an essential role of the α integrin cytoplasmic membrane-distal region in integrin activation and provide new insight of how talin and kindlin induce the high affinity integrin conformation that is required for fully functional integrins. DisclosuresNo relevant conflicts of interest to declare.

Complete integrin headpiece opening in eight steps

Carefully soaking crystals with Arg-Gly-Asp (RGD) peptides, we captured eight distinct RGD-bound conformations of the αIIbβ3 integrin headpiece. Starting from the closed βI domain conformation, we saw six intermediate βI conformations and finally the fully open βI with the hybrid domain swung out in the crystal lattice. The β1-α1 backbone that hydrogen bonds to the Asp side chain of RGD was the first element to move followed by adjacent to metal ion-dependent adhesion site Ca(2+), α1 helix, α1' helix, β6-α7 loop, α7 helix, and hybrid domain. We define in atomic detail how conformational change was transmitted over long distances in integrins, 40 Å from the ligand binding site to the opposite end of the βI domain and 80 Å to the far end of the hybrid domain. During these movements, RGD slid in its binding groove toward αIIb, and its Arg side chain became ordered. RGD concentration requirements in soaking suggested a >200-fold higher affinity after opening. The thermodynamic cycle shows how higher affinity pays the energetic cost of opening.

Modulation of integrin activation and signaling by α1/α1′-helix unbending at the junction

How conformational signals initiated from one end of the integrin are transmitted to the other end remains elusive. At the ligand-binding βI domain, the α1/α1'-helix changes from a bent to a straightened α-helical conformation upon integrin headpiece opening. We demonstrated that a conserved glycine at the α1/α1' junction is crucial for maintaining the bent conformation of the α1/α1'-helix in the resting state. Mutations that facilitate α1/α1'-helix unbending rendered integrin constitutively active; however, mutations that block the α1/α1'-helix unbending abolished soluble ligand binding upon either outside or inside stimuli. Such mutations also blocked ligand-induced integrin extension from outside the cell, but had no effect on talin-induced integrin extension from inside the cell. In addition, integrin-mediated cell spreading, F-actin stress fiber and focal adhesion formation, and focal adhesion kinase activation were also defective in these mutant integrins, although the cells still adhered to immobilized ligands at a reduced level. Our data establish the structural role of the α1/α1' junction that allows relaxation of the α1/α1'-helix in the resting state and transmission of bidirectional conformational signals by helix unbending upon integrin activation.

α(V)β(3) integrin crystal structures and their functional implications.

Many questions about the significance of structural features of integrin α(V)β(3) with respect to its mechanism of activation remain. We have determined and re-refined crystal structures of the α(V)β(3) ectodomain linked to C-terminal coiled coils (α(V)β(3)-AB) and four transmembrane (TM) residues in each subunit (α(V)β(3)-1TM), respectively. The α(V) and β(3) subunits with four and eight extracellular domains, respectively, are bent at knees between the integrin headpiece and lower legs, and the headpiece has the closed, low-affinity conformation. The structures differ in the occupancy of three metal-binding sites in the βI domain. Occupancy appears to be related to the pH of crystallization, rather than to the physiologic regulation of ligand binding at the central, metal ion-dependent adhesion site. No electron density was observed for TM residues and much of the α(V) linker. α(V)β(3)-AB and α(V)β(3)-1TM demonstrate flexibility in the linker between their extracellular and TM domains, rather than the previously proposed rigid linkage. A previously postulated interface between the α(V) and β(3) subunits at their knees was also not supported, because it lacks high-quality density, required rebuilding in α(V)β(3)-1TM, and differed markedly between α(V)β(3)-1TM and α(V)β(3)-AB. Together with the variation in domain-domain orientation within their bent ectodomains between α(V)β(3)-AB and α(V)β(3)-1TM, these findings are compatible with the requirement for large structural changes, such as extension at the knees and headpiece opening, in conveying activation signals between the extracellular ligand-binding site and the cytoplasm.

Distinct roles for talin-1 and kindlin-3 in LFA-1 extension and affinity regulation

AbstractIn inflammation, neutrophils and other leukocytes roll along the microvascular endothelium before arresting and transmigrating into inflamed tissues. Arrest requires conformational activation of the integrin lymphocyte function-associated antigen-1 (LFA-1). Mutations of the FERMT3 gene encoding kindlin-3 underlie the human immune deficiency known as leukocyte adhesion deficiency-III. Both kindlin-3 and talin-1, another FERM domain-containing cytoskeletal protein, are required for integrin activation, but their individual roles in the induction of specific integrin conformers are unclear. Here, we induce differential LFA-1 activation in neutrophils through engagement of the selectin ligand P-selectin glycoprotein ligand-1 or the chemokine receptor CXCR2. We find that talin-1 is required for inducing LFA-1 extension, which corresponds to intermediate affinity and induces neutrophil slow rolling, whereas both talin-1 and kindlin-3 are required for induction of the high-affinity conformation of LFA-1 with an open headpiece, which results in neutrophil arrest. In vivo, both slow rolling and arrest are defective in talin-1–deficient neutrophils, whereas only arrest is defective in kindlin-3–deficient neutrophils. We conclude that talin-1 and kindlin-3 serve distinct functions in LFA-1 activation.

Distinct roles for talin‐1 and kindlin‐3 in LFA‐1‐dependent neutrophil rolling and arrest

β2 integrins, including LFA‐1 (αLβ2), play critical roles in leukocyte recruitment during inflammation by binding to ICAM‐1 expressed on the endothelium. The affinity of integrin for ligand is determined by its global conformation, regulated by the binding of proteins to the integrin cytoplasmic tails. At least three different integrin affinity states have been observed: low affinity with bent ectodomain, intermediate affinity with extended ectodomain, and high affinity with extended ectodomain and open headpiece. Intermediate affinity LFA‐1 contributes to neutrophil rolling interactions with the endothelium, whereas high affinity LFA‐1 mediates neutrophil arrest. In the current study, we determined the roles for two integrin activating proteins, talin‐1 and kindlin‐3, in neutrophil rolling and arrest. Using established in vivo (intravital microscopy of post‐capillary venules) and ex vivo (microfluidic flow chamber) models, we found that talin‐1 is necessary for both LFA‐1‐dependent neutrophil rolling and arrest. In contrast, kindlin‐3 was required only for neutrophil arrest and was dispensable for rolling mediated by intermediate affinity LFA‐1. We further corroborate our results with imaging and in vitro studies using antibodies that directly report β2 integrin conformation. These data indicate that talin‐1 is sufficient to induce LFA‐1 extension to an intermediate affinity state whereas kindlin‐3 is critical for the transition of LFA‐1 to a high affinity state.

Structural specializations of α4β7, an integrin that mediates rolling adhesion

The lymphocyte homing receptor integrin α(4)β(7) is unusual for its ability to mediate both rolling and firm adhesion. α(4)β(1) and α(4)β(7) are targeted by therapeutics approved for multiple sclerosis and Crohn's disease. Here, we show by electron microscopy and crystallography how two therapeutic Fabs, a small molecule (RO0505376), and mucosal adhesion molecule-1 (MAdCAM-1) bind α(4)β(7). A long binding groove at the α(4)-β(7) interface for immunoglobulin superfamily domains differs in shape from integrin pockets that bind Arg-Gly-Asp motifs. RO0505376 mimics an Ile/Leu-Asp motif in α(4) ligands, and orients differently from Arg-Gly-Asp mimics. A novel auxiliary residue at the metal ion-dependent adhesion site in α(4)β(7) is essential for binding to MAdCAM-1 in Mg(2+) yet swings away when RO0505376 binds. A novel intermediate conformation of the α(4)β(7) headpiece binds MAdCAM-1 and supports rolling adhesion. Lack of induction of the open headpiece conformation by ligand binding enables rolling adhesion to persist until integrin activation is signaled.

Distinct Roles for Talin-1 and Kindlin-3 in Regulating LFA-1 Conformation during Neutrophil Rolling and Arrest

β2 integrins, including LFA-1 (αLβ2), play critical roles in leukocyte trafficking. LFA-1 mediates the recruitment of neutrophils during inflammation through interaction with ICAM-1 expressed on the endothelium. The affinity of integrin for ligand is determined by its global conformation, regulated by the binding of proteins to the integrin cytoplasmic tails. At least three different integrin affinity states have been observed: low affinity with bent ectodomain, intermediate affinity with extended ectodomain, and high affinity with extended ectodomain and open headpiece. Intermediate affinity LFA-1 contributes to neutrophil rolling interactions with the endothelium, whereas high affinity LFA-1 mediates neutrophil arrest. In the current study, we determined the roles for two integrin tail-binding activating proteins, talin-1 and kindlin-3, in neutrophil rolling and arrest. Using established in vivo (intravital microscopy of post-capillary venules) and ex vivo (microfluidic flow chamber) models, we found that talin-1 is necessary for both LFA-1-dependent neutrophil rolling and arrest. In contrast, kindlin-3 is required only for neutrophil arrest and is dispensable for rolling mediated by intermediate affinity LFA-1. These data indicate that talin-1 is sufficient to induce LFA-1 extension to an intermediate affinity state whereas kindlin-3 is critical for the transition of LFA-1 to a high affinity state. We further corroborated our results with imaging and in vitro studies using antibodies that directly report β2 integrin conformation. Our results are the first to demonstrate that members of the talin and kindlin protein families play distinct roles in regulating integrin conformation.

Intact αIIbβ3 Integrin Is Extended after Activation as Measured by Solution X-ray Scattering and Electron Microscopy

Integrins are bidirectional signaling molecules on the cell surface that have fundamental roles in regulating cell behavior and contribute to cell migration and adhesion. Understanding of the mechanism of integrin signaling and activation has been advanced with truncated ectodomain preparations; however, the nature of conformational change in the full-length intact integrin molecule remains an active area of research. Here we used small angle x-ray scattering and electron microscopy to study detergent-solubilized, intact platelet integrin α(IIb)β(3). In the resting state, the intact α(IIb)β(3) adopted a compact, bent conformation. Upon activation with Mn(2+), the average integrin extension increased. Further activation by addition of ligand led to stabilization of the extended state and opening of the headpiece. The observed extension and conformational rearrangement upon activation are consistent with the extension and headpiece opening model of integrin activation.