RESUMO
Integrin α5ß1 is crucial for cell attachment and migration in development and tissue regeneration, and α5ß1 binding proteins could have considerable utility in regenerative medicine and next-generation therapeutics. We use computational protein design to create de novo α5ß1-specific modulating miniprotein binders, called NeoNectins, that bind to and stabilize the open state of α5ß1. When immobilized onto titanium surfaces and throughout 3D hydrogels, the NeoNectins outperform native fibronectin and RGD peptide in enhancing cell attachment and spreading, and NeoNectin-grafted titanium implants outperformed fibronectin and RGD-grafted implants in animal models in promoting tissue integration and bone growth. NeoNectins should be broadly applicable for tissue engineering and biomedicine.
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Integrins are cell surface receptors that mediate the interactions of cells with their surroundings and play essential roles in cell adhesion, migration, and homeostasis. Eight of the 24 integrins bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, comprising the RGD-binding integrin subfamily. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands to fulfill specific functions in cellular processes. Antibodies against individual RGD-binding integrins are desirable for investigating their specific functions, and were selected here from a synthetic yeast-displayed Fab library. We discovered 11 antibodies that exhibit high specificity and affinity toward their target integrins, i.e. αVß3, αVß5, αVß6, αVß8, and α5ß1. Of these, six are function-blocking antibodies and contain a ligand-mimetic R(G/L/T)D motif in their CDR3 sequences. We report antibody-binding specificity, kinetics, and binding affinity for purified integrin ectodomains, as well as intact integrins on the cell surface. We further used these antibodies to reveal binding preferences of the αV subunit for its 5 ß-subunit partners: ß6 = ß8 > ß3 > ß1 = ß5.
Assuntos
Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Cadeias beta de Integrinas/imunologia , Cadeias beta de Integrinas/química , Cadeias beta de Integrinas/metabolismo , Cadeias beta de Integrinas/genética , Integrina alfaV/imunologia , Integrina alfaV/metabolismo , Integrinas/imunologia , Integrinas/metabolismo , Biblioteca de Peptídeos , Técnicas de Visualização da Superfície Celular , Ligação Proteica , Especificidade de AnticorposRESUMO
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 ß-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.
Assuntos
Integrinas , Talina , Animais , Humanos , Camundongos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/química , Adesão Celular , Células CHO , Cricetulus , Integrinas/metabolismo , Integrinas/química , Ligantes , Ligação Proteica , Conformação Proteica , Transdução de Sinais , Imagem Individual de Molécula , Talina/metabolismo , Talina/químicaRESUMO
Eight of the 24 integrin heterodimers bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, and play essential roles in cell adhesion, migration, and homeostasis. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands including fibronectin, vitronectin, fibrinogen, nephronectin and the prodomain of the transforming growth factors to fulfill specific functions in cellular processes. Subtype-specific antibodies against RGD-binding integrins are desirable for investigating their specific functions. In this study, we discovered 11 antibodies that exhibit high specificity and affinity towards integrins αVß3, αVß5, αVß6, αVß8, and α5ß1 from a synthetic yeast-displayed Fab library. Of these, 6 are function-blocking antibodies containing an R(G/L/T) D motif in their CDR3 sequences. We report antibody binding specificity, kinetics, and binding affinity for purified integrin ectodomains as well as intact integrins on the cell surface. We further employed these antibodies to reveal binding preferences of the αV subunit for its 5 ß-subunit partners: ß6=ß8>ß3>ß1=ß5.
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The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvß6 and αvß8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvß6 and αvß8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the αvß6 and the αvß8 integrins. In a lung fibrosis mouse model, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.
Assuntos
Integrinas , Fibrose Pulmonar , Animais , Camundongos , Membrana Celular , Microscopia Crioeletrônica , Modelos Animais de DoençasRESUMO
Microneme protein 2 (MIC2) and MIC2-associated protein (M2AP) play crucial roles in the gliding motility and host cell invasion of Toxoplasma gondii. Complex formation between MIC2 and M2AP is required for maturation and transport from the microneme to the parasite surface. Previous studies showed that M2AP associates with the 6th TSR domain of MIC2 (TSR6), but the detailed interaction remains unclear. In this study, we report crystal structures of M2AP alone and in complex with TSR6. TSR domains have an unusually thin, long structure with a layer of intercalated residues on one side. The non-layered side of TSR6 with hotspot residue His-620 at the center binds to M2AP. Remarkably, we show that TSR6 residue Y602 is dynamic; it equilibrates between being part of the layer (the layered state) and in a flipped-out state in the absence of M2AP. However, when bound to M2AP, Y602 shifts to the flipped-out state. Our findings provide insights into the association and stabilization of MIC2-M2AP complex, and may be used to develop new therapies to prevent infections caused by this parasite.
Assuntos
Toxoplasma , Humanos , Reconhecimento Psicológico , PesquisadoresRESUMO
The RGD (Arg-Gly-Asp)-binding integrins αvß6 and αvß8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between the two closely related integrin proteins and other RGD integrins, stabilize specific conformational states, and have sufficient stability enabling tissue restricted administration could have considerable therapeutic utility. Existing small molecules and antibody inhibitors do not have all of these properties, and hence there is a need for new approaches. Here we describe a method for computationally designing hyperstable RGD-containing miniproteins that are highly selective for a single RGD integrin heterodimer and conformational state, and use this strategy to design inhibitors of αvß6 and αvß8 with high selectivity. The αvß6 and αvß8 inhibitors have picomolar affinities for their targets, and >1000-fold selectivity over other RGD integrins. CryoEM structures are within 0.6-0.7Å root-mean-square deviation (RMSD) to the computational design models; the designed αvß6 inhibitor and native ligand stabilize the open conformation in contrast to the therapeutic anti-αvß6 antibody BG00011 that stabilizes the bent-closed conformation and caused on-target toxicity in patients with lung fibrosis, and the αvß8 inhibitor maintains the constitutively fixed extended-closed αvß8 conformation. In a mouse model of bleomycin-induced lung fibrosis, the αvß6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics when delivered via oropharyngeal administration mimicking inhalation, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.
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Eukaryotic cell adhesion and migration rely on surface adhesins connecting extracellular ligands to the intracellular actin cytoskeleton. Plasmodium sporozoites are transmitted by mosquitoes and rely on adhesion and gliding motility to colonize the salivary glands and to reach the liver after transmission. During gliding, the essential sporozoite adhesin TRAP engages actin filaments in the cytoplasm of the parasite, while binding ligands on the substrate through its inserted (I) domain. Crystal structures of TRAP from different Plasmodium species reveal the I domain in closed and open conformations. Here, we probe the importance of these two conformational states by generating parasites expressing versions of TRAP with the I domain stabilized in either the open or closed state with disulfide bonds. Strikingly, both mutations impact sporozoite gliding, mosquito salivary gland entry, and transmission. Absence of gliding in sporozoites expressing the open TRAP I domain can be partially rescued by adding a reducing agent. This suggests that dynamic conformational change is required for ligand binding, gliding motility, and organ invasion and hence sporozoite transmission from mosquito to mammal.
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Culicidae , Plasmodium , Animais , Esporozoítos/metabolismo , Ligantes , Plasmodium/metabolismo , Fígado/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Mamíferos/metabolismoRESUMO
Collagen superfamily of proteins is a major component of the extracellular matrix. Defects in collagens underlie the cause of nearly 40 human genetic diseases in millions of people worldwide. Pathogenesis typically involves genetic alterations of the triple helix, a hallmark structural feature that bestows exceptional mechanical resistance to tensile forces and a capacity to bind a plethora of macromolecules. Yet, there is a paramount knowledge gap in understanding the functionality of distinct sites along the triple helix. Here, we present a recombinant technique to produce triple helical fragments for functional studies. The experimental strategy utilizes the unique capacity of the NC2 heterotrimerization domain of collagen IX to drive three α-chain selection and registering the triple helix stagger. For proof of principle, we produced and characterized long triple helical fragments of collagen IV that were expressed in a mammalian system. The heterotrimeric fragments encompassed the CB3 trimeric peptide of collagen IV, which harbors the binding motifs for α1ß1 and α2ß1 integrins. Fragments were characterized and shown to have a stable triple helix, post-translational modifications, and high affinity and specific binding of integrins. The NC2 technique is a universal tool for the high-yield production of heterotrimeric fragments of collagens. Fragments are suitable for mapping functional sites, determining coding sequences of binding sites, elucidating pathogenicity and pathogenic mechanisms of genetic mutations, and production of fragments for protein replacement therapy.
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Colágeno Tipo IV , Integrinas , Multimerização Proteica , Animais , Humanos , Sítios de Ligação , Colágeno Tipo IV/química , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Integrinas/química , Integrinas/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Mutação , Domínios ProteicosRESUMO
Activation of latent transforming growth factor (TGF)-ß2 is incompletely understood. Unlike TGF-ß1 and ß3, the TGF-ß2 prodomain lacks a seven-residue RGDLXX (L/I) integrin-recognition motif and is thought not to be activated by integrins. Here, we report the surprising finding that TGF-ß2 contains a related but divergent 13-residue integrin-recognition motif (YTSGDQKTIKSTR) that specializes it for activation by integrin αVß6 but not αVß8. Both classes of motifs compete for the same binding site in αVß6. Multiple changes in the longer motif underlie its specificity. ProTGF-ß2 structures define interesting differences from proTGF-ß1 and the structural context for activation by αVß6. Some integrin-independent activation is also seen for proTGF-ß2 and even more so for proTGF-ß3. Our findings have important implications for therapeutics to αVß6 in clinical trials for fibrosis, in which inhibition of TGF-ß2 activation has not been anticipated.
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Integrinas , Fator de Crescimento Transformador beta2 , Humanos , Integrinas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Antígenos de Neoplasias/metabolismo , Fibrose , Fator de Crescimento Transformador beta/metabolismoRESUMO
Although integrins are known to be mechanosensitive and to possess many subtypes that have distinct physiological roles, single molecule studies of force exertion have thus far been limited to RGD-binding integrins. Here, we show that integrin α4ß1 and RGD-binding integrins (αVß1 and α5ß1) require markedly different tension thresholds to support cell spreading. Furthermore, actin assembled downstream of α4ß1 forms cross-linked networks in circularly spread cells, is in rapid retrograde flow, and exerts low forces from actin polymerization. In contrast, actin assembled downstream of αVß1 forms stress fibers linking focal adhesions in elongated cells, is in slow retrograde flow, and matures to exert high forces (>54-pN) via myosin II. Conformational activation of both integrins occurs below 12-pN, suggesting that post-activation subtype-specific cytoskeletal remodeling imposes the higher threshold for spreading on RGD substrates. Multiple layers of single integrin mechanics for activation, mechanotransduction and cytoskeleton remodeling revealed here may underlie subtype-dependence of diverse processes such as somite formation and durotaxis.
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Actinas , Integrina beta1 , Mecanotransdução Celular , Integrina alfa4beta1 , OligopeptídeosRESUMO
This Viewpoint discusses the rapid advances in molecular cell biological approaches over the past 50 years and the many avenues for future advances that have been opened, including direct applications for therapeutic and regenerative medicine.
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Distinções e Prêmios , Biologia Celular , Integrinas , Pesquisa Biomédica , Biologia Celular/história , Biologia Celular/tendências , História do Século XXI , Integrinas/fisiologia , Estados UnidosRESUMO
Integrins are validated drug targets with six approved therapeutics. However, small-molecule inhibitors to three integrins failed in late-stage clinical trials for chronic indications. Such unfavorable outcomes may in part be caused by partial agonism, i.e., the stabilization of the high-affinity, extended-open integrin conformation. Here, we show that the failed, small-molecule inhibitors of integrins αIIbß3 and α4ß1 stabilize the high-affinity conformation. Furthermore, we discovered a simple chemical feature present in multiple αIIbß3 antagonists that stabilizes integrins in their bent-closed conformation. Closing inhibitors contain a polar nitrogen atom that stabilizes, via hydrogen bonds, a water molecule that intervenes between a serine residue and the metal in the metal-ion-dependent adhesion site (MIDAS). Expulsion of this water is a requisite for transition to the open conformation. This change in metal coordination is general to integrins, suggesting broad applicability of the drug-design principle to the integrin family, as validated with a distantly related integrin, α4ß1.
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Desenho de Fármacos , Integrina alfa4beta1 , Conformação Proteica , Serina , ÁguaRESUMO
von Willebrand factor (VWF) is a multimeric blood protein that acts as a mechanical probe, responding to changes in flow to initiate platelet plug formation. Previously, our laboratory tests had shown that using single-molecule imaging that shear stress can extend surface-tethered VWF, but paradoxically, we found that the required shear stress was higher than reported for free-in-flow VWF, an observation inconsistent with basic physical principles. To resolve this inconsistency critical to VWF's molecular mechanism, we measured free-VWF extension in shear flow using pulsed laser stroboscopic imaging of single molecules. Here, laser pulses of different durations are used to capture multiple images of the same molecule within each frame, enabling accurate length measurements in the presence of motion blur. At high shear stresses, we observed a mean shift in VWF extension of <200 nm, much shorter than the multiple-micron extensions previously reported with no evidence for the predicted sharp globule-stretch conformational transition. Modeling VWF with a Brownian dynamics simulation, our results were consistent with VWF behaving as an uncollapsed polymer rather than the theorized compact ball. The muted response of free VWF to high shear rates implies that the tension experienced by free VWF in physiological shear flow is lower than indicated by previous reports and that tethering to platelets or the vessel wall is required to mechanically activate VWF adhesive function for primary hemostasis.
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Imagem Individual de Molécula , Fator de von WillebrandRESUMO
Integrin α5ß1 mediates cell adhesion to the extracellular matrix by binding fibronectin (Fn). Selectivity for Fn by α5ß1 is achieved through recognition of an RGD motif in the 10th type III Fn domain (Fn10) and the synergy site in the ninth type III Fn domain (Fn9). However, details of the interaction dynamics are unknown. Here, we compared synergy-site and Fn-truncation mutations for their α5ß1-binding affinities and stabilities. We also interrogated binding of the α5ß1 ectodomain headpiece fragment to Fn using hydrogen-deuterium exchange (HDX) mass spectrometry to probe binding sites and sites of integrin conformational change. Our results suggest the synergistic effect of Fn9 requires both specific residues and a folded domain. We found some residues considered important for synergy are required for stability. Additionally, we show decreases in fibronectin HDX are localized to a synergy peptide containing contacting residues in two ß-strands, an intervening loop in Fn9, and the RGD-containing loop in Fn10, indicative of binding sites. We also identified binding sites in the α5-subunit ß-propeller domain for the Fn9 synergy site and in the ß1-subunit ßI domain for Fn10 based on decreases in α5ß1 HDX. Interestingly, the dominant effect of Fn binding was an increase in α5ß1 deuterium exchange distributed over multiple sites that undergo changes in conformation or solvent accessibility and appear to be sites where energy is stored in the higher-energy, open-integrin conformation. Together, our results highlight regions important for α5ß1 binding to Fn and dynamics associated with this interaction.
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Fibronectinas , Integrina alfa5beta1 , Domínios e Motivos de Interação entre Proteínas , Sítios de Ligação , Adesão Celular , Medição da Troca de Deutério , Fibronectinas/química , Fibronectinas/genética , Integrina alfa5beta1/química , Mutação , Oligopeptídeos/química , SolventesRESUMO
Here, we study the gamete fusogen HAP2 from Cyanidioschyzon merolae (Cyani), an extremophile red algae that grows at acidic pH at 45 °C. HAP2 has a trimeric postfusion structure with similarity to viral class II fusion proteins, but its prefusion structure has been elusive. The crystal structure of a monomeric prefusion state of Cyani HAP2 shows it is highly extended with three domains in the order D2, D1, and D3. Three hydrophobic fusion loops at the tip of D2 are each required for postfusion state formation. We followed by negative stain electron microscopy steps in the process of detergent micelle-stimulated postfusion state formation. In an intermediate state, two or three linear HAP2 monomers associate at the end of D2 bearing its fusion loops. Subsequently, D2 and D1 line the core of a trimer and D3 folds back over the exterior of D1 and D2. D3 is not required for formation of intermediate or postfusion-like states.
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Extremófilos , Proteínas do Envelope Viral , Extremófilos/metabolismo , Células Germinativas/metabolismo , Conformação Proteica , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/metabolismoRESUMO
von Willebrand factor (VWF) is an adhesive glycoprotein that circulates in the blood as disulfide-linked concatemers and functions in primary hemostasis. The loss of long VWF concatemers is associated with the excessive bleeding of type 2A von Willebrand disease (VWD). Formation of the disulfide bonds that concatemerize VWF requires VWF to self-associate into helical tubules, yet how the helical tubules template intermolecular disulfide bonds is not known. Here, we report electron cryomicroscopy (cryo-EM) structures of VWF tubules before and after intermolecular disulfide bond formation. The structures provide evidence that VWF tubulates through a charge-neutralization mechanism and that the A1 domain enhances tubule length by crosslinking successive helical turns. In addition, the structures reveal disulfide states before and after disulfide bond-mediated concatemerization. The structures and proposed assembly mechanism provide a foundation to rationalize VWD-causing mutations.
Assuntos
Doença de von Willebrand Tipo 2 , Doenças de von Willebrand , Fator de von Willebrand , Dissulfetos/química , Humanos , Mutação , Domínios Proteicos , Doença de von Willebrand Tipo 2/genética , Doenças de von Willebrand/genética , Fator de von Willebrand/genéticaRESUMO
Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor (VWF) to GPIbα on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from force-induced unfolding by a long-range disulfide that links cysteines near its N- and C-termini. The O-glycosylated linkers between A1 and its neighboring domains, which transmit tensile force to A1, are reported to regulate A1 activation for binding to GPIb, but the mechanism is controversial and incompletely defined. Here, we study how these linkers, and their polypeptide and O-glycan moieties, regulate A1 affinity by measuring affinity, kinetics, thermodynamics, hydrogen deuterium exchange (HDX), and unfolding by temperature and urea. The N-linker lowers A1 affinity 40-fold with a stronger contribution from its O-glycan than polypeptide moiety. The N-linker also decreases HDX in specific regions of A1 and increases thermal stability and the energy gap between its native state and an intermediate state, which is observed in urea-induced unfolding. The C-linker also decreases affinity of A1 for GPIbα, but in contrast to the N-linker, has no significant effect on HDX or A1 stability. Among different models for A1 activation, our data are consistent with the model that the intermediate state has high affinity for GPIbα, which is induced by tensile force physiologically and regulated allosterically by the N-linker.
Assuntos
Plaquetas , Fator de von Willebrand , Plaquetas/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Ureia/metabolismo , Fator de von Willebrand/metabolismoRESUMO
TGFß has multiple roles and gene products (TGFß1, -ß2, and -ß3), which make global targeting of TGFß undesirable. Expression of TGFß requires association with milieu molecules, which localize TGFß to the surface of specific cells or extracellular matrices. Here, we found that LRRC33 was specifically associated with TGFß1, not TGFß2 and TGFß3, and was required for surface display and activation of TGFß1 on tumor-infiltrating myeloid cells. Loss of LRRC33-dependent TGFß1 activation slowed tumor growth and metastasis by enhancing innate and adaptive antitumor immunity in multiple mouse syngeneic tumor models. LRRC33 loss resulted in a more immunogenic microenvironment, with decreased myeloid-derived suppressor cells, more active CD8+ T and NK cells, and more skewing toward tumor-suppressive M1 macrophages. LRRC33 loss and PD-1 blockade synergized in controlling B16.F10 tumor growth. Our results demonstrate the importance of LRRC33 in tumor biology and highlight the therapeutic potential of dual blockade of the LRRC33/TGFß1 axis and PD-1/PD-L1 in cancer immunotherapy.
Assuntos
Imunoterapia , Neoplasias , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Imunoterapia/métodos , Macrófagos/metabolismo , Camundongos , Neoplasias/patologia , Fator de Crescimento Transformador beta/metabolismo , Microambiente TumoralRESUMO
Activation of integrins by Mn2+ is a benchmark in the integrin field, but how Mn2+ works and whether it reproduces physiological activation is unknown. We show that Mn2+ and high Mg2+ concentrations compete with Ca2+ at the ADMIDAS and shift the conformational equilibrium toward the open state, but the shift is far from complete. Additionally, replacement of Mg2+ by Mn2+ at the MIDAS increases the intrinsic affinities of both the high-affinity open and low-affinity closed states of integrins, in agreement with stronger binding of Mn2+ than Mg2+ to oxygen atoms. Mutation of the ADMIDAS increases the affinity of closed states and decreases the affinity of the open state and thus reduces the difference in affinity between the open and closed states. An important biological function of the ADMIDAS may be to stabilize integrins in highly discrete states, so that when integrins support cell adhesion and migration, their high and low affinity correspond to discrete on and off states, respectively.