Monoclonal antibodies to ligand-occupied conformers of integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) alter receptor affinity, specificity, and function

Occupancy of integrin receptors induces conformational changes in the receptor, resulting in exposure of novel interactive sites termed ligand-induced binding sites (LIBS). We report here that Fab fragments of certain antibodies against LIBS on integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) block platelet aggregation. Thus, certain LIBS or the regions surrounding them may participate in events required for platelet aggregation. In addition, certain anti-alpha IIb beta 3 LIBS Fab fragments stimulated platelet aggregation. This was due to induction of fg binding to alpha IIb beta 3, apparently by shifting a conformational equilibrium between a "resting" and an "activated" state of alpha IIb beta 3. Some of the activating anti-LIBS Fab fragments also induced high affinity fibronectin binding to alpha IIb beta 3, whereas others did not. Thus, changes in the conformation of this integrin modulate both the specificity and affinity of ligand recognition.     

Trans-dominant inhibition of integrin function.

Occupancy of integrin adhesion receptors can alter the functions of other integrins and cause partition of the ligand-occupied integrin into focal adhesions. Ligand binding also changes the conformation of integrin extracellular domains. To explore the relationship between ligand-induced conformational change and integrin signaling, we examined the effect of ligands specific for integrin alpha IIb beta 3 on the functions of target integrins alpha 5 beta 1 and alpha 2 beta 1. We report that binding of integrin-specific ligands to a suppressive integrin can inhibit the function of other target integrins (trans-dominant inhibition). Trans-dominant inhibition is due to a blockade of integrin signaling. Furthermore, this inhibition involves both a conformational change in the extracellular domain and the presence of the beta cytoplasmic tail in the suppressive integrin. Similarly, ligand-induced recruitment of alpha IIb beta 3 to focal adhesions also involves a conformational rearrangement of its extracellular domain. These findings imply that the ligand-induced conformational changes can propagate from an integrin's extracellular to its intracellular face. Trans-dominant inhibition by integrin ligands may coordinate integrin signaling and can lead to unexpected biological effects of integrin-specific inhibitors.     

A receptor-induced binding site in fibrinogen elicited by its interaction with platelet membrane glycoprotein IIb-IIIa.

The interaction of fibrinogen with membrane glycoprotein GPIIb-IIIa regulates platelet aggregation. This ligand:integrin receptor interaction elicits conformational changes in GPIIb-IIIa as evidenced by the induction of ligand-induced binding sites which are recognized by antibodies that react selectively with the occupied receptor. The dynamic nature of these conformational changes is now demonstrated by the identification and characterization of a receptor-induced binding site (RIBS) elicited in fibrinogen bound to GPIIb-IIIa. A monoclonal antibody to fibrinogen, anti-Fg-RIBS-I, failed to bind to nonstimulated platelets in the presence or absence of fibrinogen. However, when platelets were stimulated with an agonist, the antibody reacted with platelet-bound fibrinogen even in the presence of a marked excess of unbound fibrinogen. A key element of the RIBS epitope has been precisely localized to residues 373-385 of the gamma chain of fibrinogen. Conformational elements also are important in defining the epitope. Fab fragments of the antibody inhibited platelet aggregation. As these fragments also inhibited fibrin polymerization, a commonality between these two diverse functions of fibrinogen in thrombus formation is indicated. In general, antibodies to RIBS and ligand-induced binding site provide unique probes for characterizing ligand:receptor interactions.     

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.     

Probing chemical and conformational differences in the resting and active conformers of platelet integrin alpha(IIb)beta(3)

Integrin alpha(IIb)beta(3) is the fibrinogen receptor that mediates platelet adhesion and aggregation. The ligand binding function of alpha(IIb)beta(3) is "activated" on the platelet surface by physiologic stimuli. Two forms of alpha(IIb)beta(3) can be purified from platelet lysates. These forms are facsimiles of the resting (Activation State-1 or AS-1) and the active (Activation State-2 or AS-2) conformations of the integrin found on the platelet surface. Here, the differences between purified AS-1 and AS-2 were examined to gain insight into the mechanism of activation. Four major findings are put forth. 1) The association rate (k(1)) between fibrinogen and the integrin is a key difference between AS-1 and AS-2. 2) Although the divalent ion Mn(2+) enhances the ligand binding function of AS-1, this ion is unable to convert AS-1 to AS-2. Therefore, its effect on integrin is unrelated to activation. 3) Peptide mass fingerprints indicate that the chemical structure of AS-1 and AS-2 are virtually identical, calling into question the idea that post-translational modifications are necessary for activation. 4) The two forms of alpha(IIb)beta(3) have significant conformational differences at three positions. These include the junction of the heavy and light chain of alpha(IIb), the divalent ion binding sites on alpha(IIb), and at a disulfide-bonded knot linking the amino terminus of beta(3) to the cysteine-rich domain. These observations indicate that integrin is activated by a series of specific conformational rearrangements in the ectodomain that increase the rate of ligand association.     

Structures of the alpha L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation

The structure of the I domain of integrin alpha L beta 2 bound to the Ig superfamily ligand ICAM-1 reveals the open ligand binding conformation and the first example of an integrin-IgSF interface. The I domain Mg2+ directly coordinates Glu-34 of ICAM-1, and a dramatic swing of I domain residue Glu-241 enables a critical salt bridge. Liganded and unliganded structures for both high- and intermediate-affinity mutant I domains reveal that ligand binding can induce conformational change in the alpha L I domain and that allosteric signals can convert the closed conformation to intermediate or open conformations without ligand binding. Pulling down on the C-terminal alpha 7 helix with introduced disulfide bonds ratchets the beta 6-alpha 7 loop into three different positions in the closed, intermediate, and open conformations, with a progressive increase in affinity.     

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.     

L-plastin peptide activation of alpha(v)beta(3)-mediated adhesion requires integrin conformational change and actin filament disassembly

L-plastin (LPL) is a leukocyte actin binding protein previously implicated in the activation of the integrin alpha(M)beta(2) on polymorphonuclear neutrophils. To determine the role for LPL in integrin activation, K562 cell adhesion to vitronectin via alpha(v)beta(3), a well-studied model for activable integrins, was examined. Cell permeant versions of peptides based on the N-terminal sequence of LPL and the LPL headpiece domain both activated alpha(v)beta(3)-mediated adhesion. In contrast to adhesion induced by treatment with phorbol 12-myristate 13-acetate (PMA), LPL peptide-activated adhesion was independent of integrin beta(3) cytoplasmic domain tyrosines and was not inhibited by cytochalasin D. Also in contrast to PMA, LPL peptides synergized with RGD ligand or Mn(2+) for generation of a conformational change in alpha(v)beta(3) associated with the high affinity state of the integrin, as determined by binding of a ligand-induced binding site antibody. Although LPL and ligand showed synergy for ligand-induced binding site expression when actin depolymerization was inhibited by jasplakinolide, LPL peptide-induced adhesion was inhibited. Thus, both actin depolymerization and ligand-induced integrin conformational change are required for LPL peptide-induced adhesion. We hypothesize that the critical steps of increased integrin diffusion and affinity enhancement may be linked via modulation of the function of the actin binding protein L-plastin.     

Selective inhibition of integrin function by antibodies specific for ligand-occupied receptor conformers

We have hypothesized that ligand-induced binding sites (LIBS), i.e. sites expressed on cell surface receptors only after ligand binding causes the receptor to change shape, mediate subsequent biological events. To test this hypothesis, we have raised monoclonal antibodies that preferentially react with an integrin (platelet glycoprotein (GP) IIb-IIIa) after it bind Arg-Gly-Asp-containing ligands. The 13 anti-LIBS antibodies obtained define at least three distinct GPIIb-IIIa epitopes; one of these epitopes is also expressed following occupancy of another integrin, the vitronectin receptor. Certain of these LIBSs appear to mediate functions, since the antibodies that define them inhibit GPIIb-IIIa-mediated fibrin clot contraction or platelet adhesion to collagen. Nevertheless, none of the anti-LIBS antibodies inhibit binding of the primary ligand, fibrinogen. These data indicate that LIBS may mediate distinct consequences of receptor occupancy.     

Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling

How ligand binding alters integrin conformation in outside-in signaling, and how inside-out signals alter integrin affinity for ligand, have been mysterious. We address this with electron microscopy, physicochemical measurements, mutational introduction of disulfides, and ligand binding to alphaVbeta3 and alphaIIbbeta3 integrins. We show that a highly bent integrin conformation is physiological and has low affinity for biological ligands. Addition of a high affinity ligand mimetic peptide or Mn(2+) results in a switchblade-like opening to an extended structure. An outward swing of the hybrid domain at its junction with the I-like domain shows conformational change within the headpiece that is linked to ligand binding. Breakage of a C-terminal clasp between the alpha and beta subunits enhances Mn(2+)-induced unbending and ligand binding.     

Signal-transducing mechanisms involved in activation of the platelet collagen receptor integrin alpha(2)beta(1)

Evidence was obtained about the mechanism responsible for platelet integrin alpha(2)beta activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin alpha(2)beta(1) activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin alpha(2)beta(1), but those operating via glycoprotein Ib cannot. Activation of alpha(2)beta(1) induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin alpha(2)beta(1) activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin alpha(2)beta(1) activation, while at the high agonist concentrations, there would be several pathways through which integrin alpha(2)beta(1) activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (B(max)) as thrombin-induced platelets, but their affinity (K(d)) for soluble collagen was 3.7-12.7-fold lower, suggesting that activated integrin alpha(2)beta(1) induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin alpha(2)beta(1); these activated forms of integrin alpha(2)beta(1) would have different conformations that determine their ligand affinity.     

Ligand-stabilized conformational states of human beta(2) adrenergic receptor: insight into G-protein-coupled receptor activation

G-protein-coupled receptors (GPCRs) are known to exist in dynamic equilibrium between inactive- and several active-state conformations, even in the absence of a ligand. Recent experimental studies on the β₂ adrenergic receptor (β₂AR) indicate that structurally different ligands with varying efficacies trigger distinct conformational changes and stabilize different receptor conformations. We have developed a computational method to study the ligand-induced rotational orientation changes in the transmembrane helices of GPCRs. This method involves a systematic spanning of the rotational orientation of the transmembrane helices (TMs) that are in the vicinity of the ligand for predicting the helical rotations that occur on ligand binding. The predicted ligand-stabilized receptor conformations are characterized by a simultaneous lowering of the ligand binding energy and a significant gain in interhelical and receptor-ligand hydrogen bonds. Using the β₂AR as a model, we show that the receptor conformational state depends on the structure and efficacy of the ligand for a given signaling pathway. We have studied the ligand-stabilized receptor conformations of five different ligands, a full agonist, norepinephrine; a partial agonist, salbutamol; a weak partial agonist, dopamine; a very weak agonist, catechol; and an inverse agonist, ICI-115881. The predicted ligand-stabilized receptor models correlate well with the experimentally observed conformational switches in β₂AR, namely, the breaking of the ionic lock between R131^3.50 at the intracellular end of TM3 (part of the DRY motif) and E268^6.30 on TM6, and the rotamer toggle switch on W286^6.48 on TM6. In agreement with trp-bimane quenching experiments, we found that norepinephrine and dopamine break the ionic lock and engage the rotamer toggle switch, whereas salbutamol, a noncatechol partial agonist only breaks the ionic lock, and the weak agonist catechol only engages the rotamer toggle switch. Norepinephrine and dopamine occupy the same binding region, between TM3, TM5, and TM6, whereas the binding site of salbutamol is shifted toward TM4. Catechol binds deeper into the protein cavity compared to the other ligands, making contact with TM5 and TM6. A part of the catechol binding site overlaps with those of dopamine and norepinephrine but not with that of salbutamol. Virtual ligand screening on 10,060 ligands on the norepinephrine-stabilized receptor conformation shows an enrichment of 38% compared to ligand unbound receptor conformation. These results show that ligand-induced conformational changes are important for developing functionally specific drugs that will stabilize a particular receptor conformation. These studies represent the first step toward a more universally applicable computational method for studying ligand efficacy and GPCR activation.     

Tests of the extension and deadbolt models of integrin activation

Despite extensive evidence that integrin conformational changes between bent and extended conformations regulate affinity for ligands, an alternative hypothesis has been proposed in which a "deadbolt" can regulate affinity for ligand in the absence of extension. Here, we tested both the deadbolt and the extension models. According to the deadbolt model, a hairpin loop in the beta3 tail domain could act as a deadbolt to restrain the displacement of the beta3 I domain beta6-alpha7 loop and maintain integrin in the low affinity state. We found that mutating or deleting the beta3 tail domain loop has no effect on ligand binding by either alphaIIbbeta 3 or alphaVbeta3 integrins. In contrast, we found that mutations that lock integrins in the bent conformation with disulfide bonds resist inside-out activation induced by cytoplasmic domain mutation. Furthermore, we demonstrated that extension is required for accessibility to fibronectin but not smaller fragments. The data demonstrate that integrin extension is required for ligand binding during integrin inside-out signaling and that the deadbolt does not regulate integrin activation.     

Conformational regulation of the fibronectin binding and alpha 3beta 1 integrin-mediated adhesive activities of thrombospondin-1

The recognition of extracellular matrix components can be regulated by conformational changes that alter the activity of cell surface integrins. We now demonstrate that conformational regulation of the matrix glycoprotein thrombospondin-1 (TSP1) can also modulate its binding to an integrin receptor. F18 1G8 is a conformation-sensitive TSP1 antibody that binds weakly to soluble TSP1 in the presence of divalent cations. However, binding of the antibody to melanoma cells was strongly stimulated by adding exogenous TSP1 in the presence of calcium, suggesting that TSP1 undergoes a conformational change following its binding to the cell surface. This conformation was not induced by known cell surface TSP1 receptors, whereas binding of F18 was stimulated when TSP1 bound to fibronectin but not to heparin or fibrinogen. Conversely, binding of F18 to TSP1 enhanced TSP1 binding to fibronectin. Exogenous fibronectin also stimulated TSP1-dependent binding of F18 to melanoma cells. Binding of the fibronectin-TSP1 complex to melanoma cells was mediated by alpha4beta1 and alpha5beta1 integrins. Furthermore, binding to F18 or fibronectin strongly enhanced the adhesive activity of immobilized TSP1 for some cell types. This enhancement of adhesion was mediated by alpha3beta1 integrin and required that the alpha3beta1 integrin be in an active state. Fibronectin also enhanced TSP1 binding to purified alpha3beta1 integrin. Therefore, both fibronectin and the F18 antibody induce conformational changes in TSP1 that enhance the ability of TSP1 to be recognized by alpha3beta1 integrin. The conformational and functional regulation of TSP1 activity by fibronectin represents a novel mechanism for extracellular signal transduction.     

A mitogen-activated protein kinase-dependent signaling pathway in the activation of platelet integrin alpha IIbbeta3

We have recently shown that the platelet integrin alpha(IIb)beta(3) is activated by von Willebrand factor (vWF) binding to its platelet receptor, glycoprotein Ib-IX (GPIb-IX), via the protein kinase G (PKG) signaling pathway. Here we show that GPIb-IX-mediated activation of integrin alpha(IIb)beta(3) is inhibited by dominant negative mutants of Raf-1 and MEK1 in a reconstituted integrin activation model in Chinese hamster ovary (CHO) cells and that the integrin-dependent platelet aggregation induced by either vWF or low dose thrombin is inhibited by MEK inhibitors PD98059 and U0126. Thus, mitogen-activated protein kinase (MAPK) pathway is important in GPIb-IX-dependent activation of platelet integrin alpha(IIb)beta(3). Furthermore, vWF binding to GPIb-IX induces phosphorylation of Thr-202/Tyr-204 of extracellular signal-regulated kinase 2 (ERK2). GPIb-IX-induced ERK2 phosphorylation is inhibited by PKG inhibitors and enhanced by overexpression of recombinant PKG. PKG activators also induce ERK phosphorylation, indicating that activation of MAPK pathway is downstream from PKG. Thus, our data delineate a novel integrin activation pathway in which ligand binding to GPIb-IX activates PKG that stimulates MAPK pathway, leading to integrin activation.     

Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta 2 adrenergic receptor

G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that activate G protein-coupled receptors are classified as either agonists or partial agonists. To study the mechanism whereby these different classes of activating ligands modulate receptor function, we directly monitored ligand-induced conformational changes in the G protein-coupling domain of the beta(2) adrenergic receptor. Fluorescence lifetime analysis of a reporter fluorophore covalently attached to this domain revealed that, in the absence of ligands, this domain oscillates around a single detectable conformation. Binding to an antagonist does not change this conformation but does reduce the flexibility of the domain. However, when the beta(2) adrenergic receptor is bound to a full agonist, the G protein coupling domain exists in two distinct conformations. Moreover, the conformations induced by a full agonist can be distinguished from those induced by partial agonists. These results provide new insight into the structural consequence of antagonist binding and the basis of agonism and partial agonism.     

Ligand binding promotes the entropy-driven oligomerization of integrin alpha IIb beta 3

Integrin alpha(IIb)beta(3) clusters on the platelet surface after binding adhesive proteins in a process that regulates signal transduction. However, the intermolecular forces driving integrin self-association are poorly understood. This work provides new insights into integrin clustering mechanisms by demonstrating how temperature and ligand binding interact to affect the oligomeric state of alpha(IIb)beta(3). The ligand-free receptor, solubilized in thermostable octyl glucoside micelles, exhibited a cooperative transition at approximately 43 degrees C, monitored by changes in intrinsic fluorescence and circular dichroism. Both signals changed in a direction opposite to that for global unfolding, and both were diminished upon binding the fibrinogen gamma-chain ligand-mimetic peptide cHArGD. Free and bound receptors also exhibited differential sensitivity to temperature-enhanced oligomerization, as measured by dynamic light scattering, sedimentation velocity, and sedimentation equilibrium. Van't Hoff analyses of dimerization constants for alpha(IIb)beta(3) complexed with cHArGD, cRGD, or eptifibatide yielded large, favorable entropy changes partly offset by unfavorable enthalpy changes. Transmission electron microscopy showed that ligand binding and 37 degrees C incubation enhanced assembly of integrin dimers and larger oligomers linked by tail-to-tail contacts. Interpretation of these images was aided by threading models for alpha(IIb)beta(3) protomers and dimers based on the ectodomain structure of alpha(v)beta(3). We propose that entropy-favorable nonpolar interactions drive ligand-induced integrin clustering and outside-in signaling.     

Rhodocytin (aggretin) activates platelets lacking alpha(2)beta(1) integrin, glycoprotein VI, and the ligand-binding domain of glycoprotein Ibalpha

Although alpha(2)beta(1) integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from the venom of Calloselasma rhodostoma, induces platelet activation that can be blocked by monoclonal antibodies against alpha(2)beta(1) integrin. This finding suggested that clustering of alpha(2)beta(1) integrin by rhodocytin is sufficient to induce platelet activation and led to the hypothesis that collagen may activate platelets by a similar mechanism. In contrast to these findings, we provided evidence that rhodocytin does not bind to alpha(2)beta(1) integrin. Here we show that the Cre/loxP-mediated loss of beta(1) integrin on mouse platelets has no effect on rhodocytin-induced platelet activation, excluding an essential role of alpha(2)beta(1) integrin in this process. Furthermore, proteolytic cleavage of the 45-kDa N-terminal domain of glycoprotein (GP) Ibalpha either on normal or on beta(1)-null platelets had no significant effect on rhodocytin-induced platelet activation. Moreover, mouse platelets lacking both alpha(2)beta(1) integrin and the activating collagen receptor GPVI responded normally to rhodocytin. Finally, even after additional proteolytic removal of the 45-kDa N-terminal domain of GPIbalpha rhodocytin induced aggregation of these platelets. These results demonstrate that rhodocytin induces platelet activation by mechanisms that are fundamentally different from those induced by collagen.     

Crystal structure of EMS16 in complex with the integrin alpha2-I domain

Snake venoms contain a number of heterodimeric C-type lectin-like proteins (CLPs) that interact specifically with components of the haemostatic system. EMS16 from the venom of Echis multisquamatus binds to the collagen receptor, integrin alpha2beta1, also known as glycoprotein (GP) Ia/IIa, and specifically inhibits collagen binding. Here we report the crystal structure of EMS16 in complex with recombinant integrin alpha2-I domain that plays a central role in collagen binding. The structure of the complex at 1.9 Angstrom resolution reveals that the collagen-binding site of the alpha2-I domain is covered completely by the bound EMS16. This blockage by EMS16 appears to spatially inhibit collagen binding to the alpha2-I domain. The bound alpha2-I domain adopts a closed conformation, which is seen in the absence of ligand, suggesting that EMS16 stabilizes a closed conformation corresponding to the less active structure of the alpha2-I domain. EMS16 does not directly bind to the manganese ion and residues of the metal ion-dependent adhesion site (MIDAS) of the alpha2-I domain, suggesting that EMS16 may have the potential to bind specifically to the alpha2-I domain in a metal ion-independent fashion.