Transition from rolling to firm adhesion is regulated by the conformation of the I domain of the integrin lymphocyte function-associated antigen-1

The integrin lymphocyte function-associated antigen-1 (alpha(L)beta(2)), which is known for its ability to mediate firm adhesion and migration, can also contribute to tethering and rolling in shear flow. The alpha(L) I domain can be mutationally locked with disulfide bonds into two distinct conformations, open and closed, which have high and low affinity for the ligand intercellular adhesion molecule 1 (ICAM-1), respectively. The wild type I domain exists primarily in the lower energy closed conformation. We have measured for the first time the effect of conformational change on adhesive behavior in shear flow. We show that wild type and locked open I domains, expressed in alpha(L)beta(2) heterodimers or as isolated domains on the cell surface, mediate rolling adhesion and firm adhesion, respectively. alpha(L)beta(2) is thus poised for the conversion of rolling to firm adhesion upon integrin activation in vivo. Isolated I domains are surprisingly more effective than alpha(L)beta(2) in interactions in shear flow, which may in part be a consequence of the presence of alpha(L)beta(2) in a bent conformation. Furthermore, the force exerted on the C-terminal alpha-helix appears to stabilize the open conformation of the wild type isolated I domain and contribute to its robustness in supporting rolling. An allosteric small molecule antagonist of alpha(L)beta(2) inhibits both rolling adhesion and firm adhesion, which has important implications for its mode of action in vivo.     

Dual function for a unique site within the beta2I domain of integrin alphaMbeta2

Integrin activation has been postulated to occur in part via conformational changes in the I domain of the beta subunit (the betaI domain), especially near the F-alpha(7) loop, in response to "inside-out" signaling. However, direct evidence for a role of the F-alpha(7) loop in ligand binding and activity modulation is still lacking. Here, we report our finding that the F-alpha(7) loop (residues 344-358) within the beta(2)I domain has dual functions in ligand binding by alpha(M)beta(2). On the one hand, it supports intercellular adhesion molecule 1 (ICAM-1) binding to alpha(M)beta(2) directly as part of a recognition interface formed by five noncontiguous segments (Pro(192)-Glu(197), Asn(213)-Glu(220), Leu(225)-Leu(230), Ser(324)-Thr(329), and Glu(344)-Asp(348)) on the apex of the beta(2)I domain. On the other hand, it controls the open and closed conformation of the alpha(M)beta(2) receptor, thereby indirectly affecting alpha(M)beta(2) binding to other ligands. Switching the five constituent sequences of the ICAM-1-binding site within the beta(2)I domain to their beta(1) counterparts destroyed ICAM-1 binding but had no effect on the gross conformations of the receptor. Of the five ICAM-1 binding-defective mutants, four had normal or even stronger interaction with Fg and C3bi, as reported in our previous study. Synthetic peptides derived from the identified site inhibited alpha(M)beta(2)-ICAM-1 interaction and supported direct binding to ICAM-1. Most importantly, perturbation of the F-alpha(7) loop caused conformational changes within the beta(2)I domain, which was further propagated to other regions of alpha(M)beta(2). Altogether, our data demonstrate that inside-out signaling could modulate ligand binding directly by changing the ligand-binding pocket per se and/or indirectly by inducing multiple conformational changes within the receptor.     

Intercellular adhesion molecule-3 binding of integrin alphaL beta2 requires both extension and opening of the integrin headpiece

The leukocyte-restricted integrin alpha(L)beta(2) is required in immune processes such as leukocyte adhesion, migration, and immune synapse formation. Activation of alpha(L)beta(2) by conformational changes promotes alpha(L)beta(2) binding to its ligands, ICAMs. It was reported that different affinity states of alpha(L)beta(2) are required for binding ICAM-1 and ICAM-3. Recently, the bent, extended with a closed headpiece, and extended with open headpiece conformations of alpha(L)beta(2), was reported. To address the overall conformational requirements of alpha(L)beta(2) that allow selective binding of these ICAMs, we examined the adhesion properties of these alpha(L)beta(2) conformers. alpha(L)beta(2) with different conformations were generated by mutations, and verified by using a panel of reporter mAbs that detect alpha(L)beta(2) extension, hybrid domain movement, or I-like domain activation. We report a marked difference between extended alpha(L)beta(2) with closed and open headpieces in their adhesive properties to ICAM-1 and ICAM-3. Our data show that the extension of alpha(L)beta(2) alone is sufficient to mediate ICAM-1 adhesion. By contrast, an extended alpha(L)beta(2) with an open headpiece is required for ICAM-3 adhesion.     

Two synergistic activation mechanisms of alpha2beta1 integrin-mediated collagen binding

Activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA) induces ligand-independent aggregation of a cell surface collagen receptor, alpha2beta1 integrin. Concomitantly, TPA increases the avidity of alpha2beta1 for collagen and the number of conformationally activated alpha2beta1 integrins. The structural change was shown using a monoclonal antibody 12F1 that recognizes the "open" (active) conformation of the inserted domain in the alpha2 subunit (alpha2I). Amino acid residue Glu-336 in alpha2 subunit is proposed to mediate the interaction between alpha2I domain and beta1 subunit. Glu-336 seems to regulate a switch between open and "closed" conformations, since the mutation alpha2E336A inhibited the TPA-related increase in the number of 12F1 positive integrins. E336A also reduced cell adhesion to collagen. However, E336A did not prevent the TPA-related increase in adhesion to collagen or alpha2beta1 aggregation. Thus, alpha2beta1 integrin avidity is regulated by two synergistic mechanisms, first an alpha2E336-dependent switch to the open alpha2I conformation, and second an alpha2E336-independent mechanism temporally associated with receptor aggregation.     

Epitope mapping of monoclonal antibody to integrin alphaL beta2 hybrid domain suggests different requirements of affinity states for intercellular adhesion molecules (ICAM)-1 and ICAM-3 binding

Integrin undergoes different activation states by changing its quaternary conformation. The integrin beta hybrid domain acts as a lever for the transmission of activation signal. The displacement of the hybrid domain can serve to report different integrin activation states. The monoclonal antibody (mAb) MEM148 is a reporter antibody that recognizes Mg/EGTA-activated but not resting integrin alpha(L) beta2. Herein, we mapped its epitope to the critical residue Pro374 located on the inner face of the beta2 hybrid domain. Integrin alpha(L) beta2 binds to its ligands ICAM-1 and ICAM-3 with different affinities. Integrin is proposed to have at least three affinity states, and the position of the hybrid domain differs in each. We made use of the property of mAb MEM148 to analyze and correlate these affinity states in regard to alpha(L) beta2/intercellular adhesion molecule (ICAM) binding. Our study showed that Mg/EGTA-activated alpha(L)beta2 can adopt a different conformation from that activated by activating mAbs KIM185 or MEM48. Unlike ICAM-1 binding, which required only one activating agent, alpha(L) beta2/ICAM-3 binding required both Mg/EGTA and an activating mAb. This suggests that alpha(L)beta2 with intermediate affinity is sufficient to bind ICAM-1 but not ICAM-3, which requires a high affinity state. Furthermore, we showed that the conformation adopted by alpha(L)beta2 in the presence of Mg/EGTA, depicting an intermediate activation state, could be reverted to its resting conformation.     

Conversion between three conformational states of integrin I domains with a C-terminal pull spring studied with molecular dynamics

We test with molecular dynamics the hypothesis that interdomain forces in integrins, simulated with a spring attached to the C-terminal alpha 7-helix of an integrin I domain, can allosterically stabilize alternative I domain conformations. Depending on the force applied and timecourse, in alpha(L) and alpha(M) I domains the beta 6-alpha 7 loop moves successively between three ratchet positions; i.e. from closed to intermediate, and then to open. More distal, linked alterations in MIDAS loops and metal coordination closely resemble those seen when the MIDAS becomes ligated. Simulations show that the intermediate state is populated over a wider range of forces for alpha(L) than alpha(M) I domains. Simulations with mutant I domains suggest that specific ratchet residues regulate conformational equilibria. Simulations with alpha(1) and alpha(2) I domains reveal a lack of the intermediate conformation, owing to Phe to Glu substitution at the second ratchet residue. The findings have important implications for biological regulation of integrin adhesiveness.     

Activation-induced conformational changes in the I domain region of lymphocyte function-associated antigen 1.

Conformational changes in integrins are important for efficient ligand binding during activation. We proposed that the I domain of the integrin lymphocyte function-associated antigen 1 (LFA-1) could exist in both open and closed conformations and generated constitutively activated LFA-1 by locking the I domain in the open conformation. Here we provide structural and biochemical evidence to validate conformational change in the I domain of LFA-1 upon activation. Two monoclonal antibodies to alpha(L), HI111 and CBR LFA-1/1, bind wild-type LFA-1 well, but their binding is significantly reduced when LFA-1 is locked in the open conformation. Furthermore, this reduction in monoclonal antibody binding also occurs when LFA-1 is activated by divalent cations. HI111 maps to the top region of the I domain that is close to the putative ligand-binding site surrounding the MIDAS (metal ion-dependent adhesion site). The epitope of CBR LFA-1/1 is at the C-terminal segment of the I domain that links to the beta-propeller, and undergoes a large movement between the open and closed conformations. Our data demonstrate that these two regions undergo significant conformational changes during LFA-1 activation and that the I domain of activated LFA-1 adopts a similar tertiary structure as the predicted locked open form.     

Transition from rolling to firm adhesion can be mimicked by extension of integrin alphaLbeta2 in an intermediate affinity state

AlphaLbeta2 affinity for intercellular adhesion molecule-1 (ICAM-1) is regulated by the conformation of the alphaL I domain, which is in turn controlled by the conformation and orientation of other adjacent domains. Additionally, overall integrin conformation (bent versus straightened) influences the orientation of the I domain and access to its ligands, influencing adhesive efficiency. The open or high affinity I domain conformation supports strong adhesion, whereas the closed, low affinity conformation mediates weak interactions or rolling. We have previously suggested that alphaLbeta2 can also exist on the cell surface in an intermediate affinity state. Here we have studied the adhesive properties of integrin alphaLbeta2 containing mutant I domains with intermediate affinities for ICAM-1. In an overall bent conformation, the intermediate affinity state of alphaLbeta2 is hardly detected by conventional adhesion assays, but robust adhesion is seen when an extended conformation is induced by a small molecule alpha/beta I allosteric antagonist. Intermediate affinity alphaLbeta2 supports more stable rolling than wild-type alphaLbeta2 under shear conditions. Moreover, antagonist-induced extension transforms rolling adhesion into firm adhesion in a manner reminiscent of chemokine activation of integrin alphaLbeta2. These findings suggest the relevance of intermediate affinity states of alphaLbeta2 to the transition between inactive and active states and demonstrate the importance of both I domain affinity and overall integrin conformation for cell adhesion.     

Locking the beta3 integrin I-like domain into high and low affinity conformations with disulfides

Although integrin alpha subunit I domains exist in multiple conformations, it is controversial whether integrin beta subunit I-like domains undergo structurally analogous movements of the alpha7-helix that are linked to affinity for ligand. Disulfide bonds were introduced into the beta(3) integrin I-like domain to lock its beta6-alpha7 loop and alpha7-helix in two distinct conformations. Soluble ligand binding, ligand mimetic mAb binding and cell adhesion studies showed that disulfide-bonded receptor alpha(IIb)beta(3)(T329C/A347C) was locked in a low affinity state, and dithiothreitol treatment restored the capability of being activated to high affinity binding; by contrast, disulfide-bonded alpha(IIb)beta(3)(V332C/M335C) was locked in a high affinity state. The results suggest that activation of the beta subunit I-like domain is analogous to that of the alpha subunit I domain, i.e. that axial movement in the C-terminal direction of the alpha7-helix is linked to rearrangement of the I-like domain metal ion-dependent adhesion site into a high affinity conformation.     

Importance of force linkage in mechanochemistry of adhesion receptors

The alpha subunit-inserted (I) domain of integrin alphaLbeta2 [lymphocyte function-associated antigen-1 (LFA-1)] binds to intercellular adhesion molecule-1 (ICAM-1). The C- and N-termini of the alpha I domain are near one another on the "lower" face, opposite the metal ion-dependent adhesion site (MIDAS) on the "upper face". In conversion to the open alpha I domain conformation, a 7 A downward, axial displacement of C-terminal helix alpha7 is allosterically linked to rearrangement of the MIDAS into its high-affinity conformation. Here, we test the hypothesis that when an applied force is appropriately linked to conformational change, the conformational change can stabilize adhesive interactions that resist the applied force. Integrin alpha I domains were anchored to the cell surface through their C- or N-termini using type I or II transmembrane domains, respectively. C-terminal but not N-terminal anchorage robustly supported cell rolling on ICAM-1 substrates in shear flow. In contrast, when the alphaL I domain was mutationally stabilized in the open conformation with a disulfide bond, it mediated comparable levels of firm adhesion with type I and type II membrane anchors. To exclude other effects as the source of differential adhesion, these results were replicated using alpha I domains conjugated through the N- or C-terminus to polystyrene microspheres. Our results demonstrate a mechanical feedback system for regulating the strength of an adhesive bond. A review of crystal structures of integrin alpha and beta subunit I domains and selectins in high- and low-affinity conformations demonstrates a common mechanochemical design in which biologically applied tensile force stabilizes the more extended, high-affinity conformation.     

The role of the CPNKEKEC sequence in the beta(2) subunit I domain in regulation of integrin alpha(L)beta(2) (LFA-1)

The alpha(L) I (inserted or interactive) domain of integrin alpha(L)beta(2) undergoes conformational changes upon activation. Recent studies show that the isolated, activated alpha(L) I domain is sufficient for strong ligand binding, suggesting the beta(2) subunit to be only indirectly involved. It has been unclear whether the activity of the alpha(L) I domain is regulated by the beta(2) subunit. In this study, we demonstrate that swapping the disulfide-linked CPNKEKEC sequence (residues 169-176) in the beta(2) I domain with a corresponding beta(3) sequence, or mutating Lys(174) to Thr, constitutively activates alpha(L)beta(2) binding to ICAM-1. These mutants do not require Mn(2+) for ICAM-1 binding and are insensitive to the inhibitory effect of Ca(2+). We have also localized a component of the mAb 24 epitope (a reporter of beta(2) integrin activation) in the CPNKEKEC sequence. Glu(173) and Glu(175) of the beta(2) I domain are identified as critical for mAb 24 binding. Because the epitope is highly expressed upon beta(2) integrin activation, it is likely that the CPNKEKEC sequence is exposed or undergoes conformational changes upon activation. Deletion of the alpha(L) I domain did not eliminate the mAb 24 epitope. This confirms that the alpha(L) I domain is not critical for mAb 24 binding, and indicates that mAb 24 detects a change expressed in part in the beta(2) subunit I domain. These results suggest that the CPNKEKEC sequence of the beta(2) I domain is involved in regulating the alpha(L) I domain.     

The leukocyte integrin alpha D beta 2 binds VCAM-1: evidence for a binding interface between I domain and VCAM-1.

The trafficking of leukocytes through tissues is supported by an interaction between the beta 2 (CD18) integrins CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) and their ligand ICAM-1. The most recently identified and fourth member of the beta 2 integrins, alpha D beta 2, selectively binds ICAM-3 and does not appear to bind ICAM-1. We have reported recently that alpha D beta 2 can support eosinophil adhesion to VCAM-1. Here we demonstrate that expression of alpha D beta 2 in a lymphoid cell that does not express alpha 4 integrins confers efficient binding to VCAM-1. In addition, a soluble form of alpha D beta 2 binds VCAM-1 with greater efficiency relative to ICAM-3. The I domain of alpha D contains a binding site for VCAM-1 since recombinant alpha D I domain binds specifically to VCAM-1. In addition, alpha D mAb that block cellular binding to VCAM-1 bind the alpha D I domain. Using VCAM-1 mutants we have determined that the binding site on VCAM-1 for alpha D beta 2 overlaps with that of alpha 4++ integrins. Substitution of VCAM-1 aspartate at position 40, D40, within the conserved integrin binding site, diminishes binding to alpha D beta 2 and abrogates binding to the alpha D I domain. The corresponding integrin binding site residue in ICAM-3 is also essential to alpha D beta 2 binding. Finally, we demonstrate that alpha D beta 2 can support lymphoid cell adhesion to VCAM-1 under flow conditions at levels equivalent to those mediated by alpha 4 beta 1. These results indicate that VCAM-1 can bind to an I domain and that the binding of alpha D beta 2 to VCAM-1 may contribute to the trafficking of a subpopulation of leukocytes that express alpha D beta 2.     

Ultrastructure and function of dimeric, soluble intercellular adhesion molecule-1 (ICAM-1)

Previous studies have demonstrated dimerization of intercellular adhesion molecule-1 (ICAM-1) on the cell surface and suggested a role for immunoglobulin superfamily domain 5 and/or the transmembrane domain in mediating such dimerization. Crystallization studies suggest that domain 1 may also mediate dimerization. ICAM-1 binds through domain 1 to the I domain of the integrin alpha(L)beta(2) (lymphocyte function-associated antigen 1). Soluble C-terminally dimerized ICAM-1 was made by replacing the transmembrane and cytoplasmic domains with an alpha-helical coiled coil. Electron microscopy revealed C-terminal dimers that were straight, slightly bent, and sometimes U-shaped. A small number of apparently closed ring-like dimers and W-shaped tetramers were found. To capture ICAM-1 dimerized at the crystallographically defined dimer interface in domain 1, cysteines were introduced into this interface. Several of these mutations resulted in the formation of soluble disulfide-bonded ICAM-1 dimers (domain 1 dimers). Combining a domain 1 cysteine mutation with the C-terminal dimers (domain 1/C-terminal dimers) resulted in significant amounts of both closed ring-like dimers and W-shaped tetramers. Surface plasmon resonance studies showed that all of the dimeric forms of ICAM-1 (domain 1, C-terminal, and domain 1/C-terminal dimers) bound similarly to the integrin alpha(L)beta(2) I domain, with affinities approximately 1.5--3-fold greater than that of monomeric ICAM-1. These studies demonstrate that ICAM-1 can form at least three different topologies and that dimerization at domain 1 does not interfere with binding in domain 1 to alpha(L)beta(2).     

Effects of conformational activation of integrin alpha 1I and alpha 2I domains on selective recognition of laminin and collagen subtypes

Collagen receptor integrins alpha 1 beta 1 and alpha 2 beta 1 can selectively recognize different collagen subtypes. Here we show that their alpha I domains can discriminate between laminin isoforms as well: alpha 1I and alpha 2I recognized laminin-111, -211 and -511, whereas their binding to laminin-411 was negligible. Residue Arg-218 in alpha1 was found to be instrumental in high-avidity binding. The gain-of-function mutation E318W makes the alpha 2I domain to adopt the "open" high-affinity conformation, while the wild-type alpha 2I domain favors the "closed" low-affinity conformation. The E318W mutation markedly increased alpha 2I domain binding to the laminins (-111, -211 and -511), leading us to propose that the activation state of the alpha 2 beta 1 integrin defines its role as a laminin receptor. However, neither wild-type nor alpha 2IE318W domain could bind to laminin-411. alpha 2IE318W also bound tighter to all collagens than alpha 2I wild-type, but it showed reduced ability to discriminate between collagens I, IV and IX. The corresponding mutation, E317A, in the alpha 1I domain transformed the domain into a high-avidity binder of collagens I and IV. Thus, our results indicate that conformational activation of integrin alpha 1I and alpha 2I domains leads to high-avidity binding to otherwise disfavored collagen subtypes.     

Computational design of an integrin I domain stabilized in the open high affinity conformation

We have taken a computational approach to design mutations that stabilize a large protein domain of approximately 200 residues in two alternative conformations. Mutations in the hydrophobic core of the alphaMbeta2 integrin I domain were designed to stabilize the crystallographically defined open or closed conformers. When expressed on the cell surface as part of the intact heterodimeric receptor, binding of the designed open and closed I domains to the ligand iC3b, a form of the complement component C3, was either increased or decreased, respectively, compared to wild type. Moreover, when expressed in isolation from other integrin domains using an artificial transmembrane domain, designed open I domains were active in ligand binding, whereas designed closed and wild type I domains were inactive. Comparison to a human expert designed open mutant showed that the computationally designed mutants are far more active. Thus, computational design can be used to stabilize a molecule in a desired conformation, and conformational change in the I domain is physiologically relevant to regulation of ligand binding.     

The binding sites for competitive antagonistic, allosteric antagonistic, and agonistic antibodies to the I domain of integrin LFA-1

We explore the binding sites for mAbs to the alpha I domain of the integrin alphaLbeta2 that can competitively inhibit, allosterically inhibit, or activate binding to the ligand ICAM-1. Ten mAbs, some of them clinically important, were mapped to species-specific residues. The results are interpreted with independent structures of the alphaL I domain determined in seven different crystal lattices and in solution, and which are present in three conformational states that differ in affinity for ligand. Six mAbs bind to adjacent regions of the beta1-alpha1 and alpha3-alpha4 loops, which show only small (mean, 0.8 angstroms; maximum, 1.8 angstroms) displacements among the eight I domain structures. Proximity to the ligand binding site and to noncontacting portions of the ICAM-1 molecule explains competitive inhibition by these mAbs. Three mAbs bind to a segment of seven residues in the beta5-alpha6 loop and alpha6 helix, in similar proximity to the ligand binding site, but on the side opposite from the beta1-alpha1/alpha3-alpha4 epitopes, and far from noncontacting portions of ICAM-1. These residues show large displacements among the eight structures in response to lattice contacts (mean, 3.6 angstroms; maximum, 9.4 angstroms), and movement of a buried Phe in the beta5-alpha6 loop is partially correlated with affinity change at the ligand binding site. Together with a lack of proximity to noncontacting portions of ICAM-1, these observations explain variation among this group of mAbs, which can either act as competitive or allosteric antagonists. One agonistic mAb binds distant from the ligand binding site of the I domain, to residues that show little movement (mean, 0.5 angstroms; maximum, 1.0 angstroms). Agonism by this mAb is thus likely to result from altering the orientation of the I domain with respect to other domains within an intact integrin alphaLbeta2 heterodimer.     

An unusual allosteric mobility of the C-terminal helix of a high-affinity alphaL integrin I domain variant bound to ICAM-5

Integrins are cell surface receptors that transduce signals bidirectionally across the plasma membrane. The key event of integrin signaling is the allosteric regulation between its ligand-binding site and the C-terminal helix (alpha7) of integrin's inserted (I) domain. A significant axial movement of the alpha7 helix is associated with the open, active conformation of integrins. We describe the crystal structure of an engineered high-affinity I domain from the integrin alpha(L)beta(2) (LFA-1) alpha subunit in complex with the N-terminal two domains of ICAM-5, an adhesion molecule expressed in telencephalic neurons. The finding that the alpha7 helix swings out and inserts into a neighboring I domain in an upside-down orientation in the crystals implies an intrinsically unusual mobility of this helix. This remarkable feature allows the alpha7 helix to trigger integrin's large-scale conformational changes with little energy penalty. It serves as a mechanistic example of how a weakly bound adhesion molecule works in signaling.     

Jararhagin-derived RKKH peptides induce structural changes in alpha1I domain of human integrin alpha1beta1

Integrin alpha(1)beta(1) is one of four collagen-binding integrins in humans. Collagens bind to the alphaI domain and in the case of alpha(2)I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In alpha(2)I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligand-bound "open" conformation of alpha(2)I differs significantly from the "closed" conformation seen in the structure of apo-alpha(2)I near MIDAS. Here we show that two peptides, CTRKKHDC and CARKKHDC, derived from jararhagin also bind to alpha(1)I and competitively inhibit collagen I binding. Furthermore, calorimetric and fluorimetric measurements show that the structure of the complex of alpha(1)I with Mg(2+) and CTRKKHDC differs from structure in the absence of peptide. A comparison of the x-ray structure of apo-alpha(1)I ("closed" conformation) and a model structure of the alpha(1)I ("open" conformation) based on the closely related structure of alpha(2)I reveals that the binding site is partially blocked to ligands by Glu(255) and Tyr(285) in the "closed" structure, whereas in the "open" structure helix C is unwound and these residues are shifted, and the "RKKH" peptides fit well when docked. The "open" conformation of alpha(2)I resulting from binding a collagen (I)-like peptide leads to exposure of hydrophobic surface, also seen in the model of alpha(1)I and shown experimentally for alpha(1)I using a fluorescent hydrophobic probe.     

A 50-A separation of the integrin alpha v beta 3 extracellular domain C termini reveals an intermediate activation state

The integrin alpha(v)beta(3) has been shown to exist in low and high affinity conformations. Activation to the high affinity state is thought to depend on the "switchblade-like" opening, from a low affinity bent conformation with a closed headpiece to an extended form of the integrin with an open headpiece. Activation has been shown to depend on separation of the cytoplasmic domains. How cytoplasmic domain separation is related to separation of the transmembrane domains is unknown, and the distance of separation of the transmembrane domains required for activation has not been defined. A constrained secreted form of alpha(v)beta(3) was engineered that introduced a 50-A separation of the integrin C-terminal tails of the extracellular domains of the alpha(v) and beta(3) subunits. Receptor binding and recognition by ligand-induced binding state (LIBS) monoclonal antibodies demonstrated that the mutant receptor was locked into a low affinity state that was likely in a partially extended conformation but with a closed headpiece. In the presence of RGD peptide, the constrained receptor was able to fully extend, as determined by full exposure of LIBS epitopes. In the presence of the appropriate LIBS antibody, high affinity ligand binding of the constrained receptor was achieved. The results support the existence of transient intermediate activation states of secreted alpha(v)beta(3). Furthermore, these results with the secreted alpha(v)beta(3) receptor support a model for the full-length membrane-bound form of alpha(v)beta(3), whereby a 50-A lateral separation of the integrin alpha(v) and beta(3) transmembrane domains would be sufficient to enforce the switchblade-like opening to the extended conformation but insufficient for full receptor activation.     

Model of the alphaLbeta2 integrin I-domain/ICAM-1 DI interface suggests that subtle changes in loop orientation determine ligand specificity

The interaction of the alphaLbeta2 integrin with its cellular ligand the intercellular adhesion molecule-1 (ICAM-1) is critical for the tight binding interaction between most leukocytes and the vascular endothelium before transendothelial migration to the sites of inflammation. In this article we have modeled the alphaL subunit I-domain in its active form, which was computationally docked with the D1 domain of the ICAM-1 to probe potential protein-protein interactions. The experimentally observed key interaction between the carboxylate of Glu 34 in the ICAM-1 D1 domain and the metal ion-dependent adhesion site (MIDAS) in the open alphaL I-domain was consistently reproduced by our calculations. The calculations reveal the nature of the alphaLbeta2/ICAM-1 interaction and suggest an explanation for the increased ligand-binding affinity in the "open" versus the "closed" conformation of the alphaL I-domain. A mechanism for substrate selectivity among alphaL, alphaM, and alpha2 I-domains is suggested whereby the orientation of the loops within the I-domain is critical in mediating the interaction of the Glu 34 carboxylate of ICAM-1 D1 with the MIDAS.