Specificity-determining Loop Research Articles

Structural insights into the molecular recognition of integrin αVβ3 by RGD-containing ligands: The role of the specificity-determining loop (SDL).

Integrin αVβ3 is a prominent member of the "RGD-recognizing" integrin family of cell surface receptors. αVβ3 binds to various extracellular matrix (ECM) proteins and oxysterols such as 25-hydroxycholesterol, is implicated in several diseases, including cancer metastasis, lung fibrosis, inflammation, and autoimmune diseases, and is pursued as a valuable therapeutic target. Despite enormous efforts to seek a pure antagonist, to date, no single drug candidate has successfully reached clinics due to associated partial agonism and toxicity issues. Developing effective and safe inhibitors require a thorough understanding of the molecular interactions and structural changes related to the receptor's activation and inhibition mechanisms. This study offers a comprehensive residue-residue contact and network analyses of the ligand-binding β-propeller βI domains (headpiece) based on all available experimental structures of integrin αVβ3 in unliganded, agonist-, antagonist-, and antibody-bound states. The analyses reveal many critical interactions that were not reported before and show that specific orientation and interactions of residues from the specificity-determining loop (SDL) are critical in molecular recognition and regulation. Also, the network analysis reveals that residues from the nearby allosteric site (site II) connect to the primary RGD-binding site via SDL, which likely acts as an interface between the two sites. Our results provide valuable insights into molecular interactions, structural changes, distinct features of the active and inactive headpiece conformations, the role of SDL in ligand recognition, and SDL-mediated allostery. Thus, the insights from this study may facilitate the designing of pure antagonists or site II-mediated allosteric modulators to integrin αVβ3 to treat various diseases.

Molecular basis for the recognition of 24-(S)-hydroxycholesterol by integrin αvβ3

A growing body of evidence suggests that oxysterols such as 25-hydroxycholesterol (25HC) are biologically active and involved in many physiological and pathological processes. Our previous study demonstrated that 25HC induces an innate immune response during viral infections by activating the integrin-focal adhesion kinase (FAK) pathway. 25HC produced the proinflammatory response by binding directly to integrins at a novel binding site (site II) and triggering the production of proinflammatory mediators such as tumor necrosis factor-α (TNF) and interleukin-6 (IL-6). 24-(S)-hydroxycholesterol (24HC), a structural isomer of 25HC, plays a critical role in cholesterol homeostasis in the human brain and is implicated in multiple inflammatory conditions, including Alzheimer’s disease. However, whether 24HC can induce a proinflammatory response like 25HC in non-neuronal cells has not been studied and remains unknown. The aim of this study was to examine whether 24HC produces such an immune response using in silico and in vitro experiments. Our results indicate that despite being a structural isomer of 25HC, 24HC binds at site II in a distinct binding mode, engages in varied residue interactions, and produces significant conformational changes in the specificity-determining loop (SDL). In addition, our surface plasmon resonance (SPR) study reveals that 24HC could directly bind to integrin αvβ3, with a binding affinity three-fold lower than 25HC. Furthermore, our in vitro studies with macrophages support the involvement of FAK and NFκB signaling pathways in triggering 24HC-mediated production of TNF. Thus, we have identified 24HC as another oxysterol that binds to integrin αvβ3 and promotes a proinflammatory response via the integrin-FAK-NFκB pathway.

Structural basis for the molecular recognition of various RGD-containing ligands by integrin αVβ3: The role of the specificity-determining loop (SDL).

Integrins are multifunctional cell surface receptors that play critical roles in normal physiological processes such as proliferation, survival, migration, and cell fate transitions. Integrins are implicated in several pathological conditions, including inflammation, tumor metastasis, lung fibrosis, and autoimmune disorders. Recent studies show that apart from their endogenous ligands, αVβ3 and α5β1 integrins bind directly to the hydroxylated form of cholesterol (25HC and 24HC) to upregulate pro-inflammatory response. Among the integrins, αVβ3 integrin has been considered a viable target for treating many diseases. Despite enormous efforts, currently available RGD-based anti-integrin drugs lack specificity, exhibit partial agonism, and cause serious side effects, including tumor growth and angiogenesis. The development of safe and efficient inhibitors requires a thorough understanding of the structural basis for outside-in activation and inhibition mechanism of integrin αvβ3. In this study, we performed a comprehensive residue-residue contact and network analysis of the αvβ3 integrin headpiece using all available experimental structures. Surprisingly, our analyses show that specific orientation and interactions of residues from the specificity-determining loop (SDL) are critical in the molecular recognition and regulation of integrin αvβ3. In addition, network analysis reveals that residues from the allosteric site (site II) connect to the primary RGD-binding site via SDL, which acts as an interface for the allosteric modulation. The obtained results provide a comprehensive structural perspective, useful insights into the distinct features of active and inactive conformations, and the role of SDL in the ligand recognition process and allostery.

Molecular insight into the outside‐in activation of integrin αvβ3 by 24( S )‐hydroxycholesterol

Objective Oxysterols such as 25-hydroxycholesterol (25HC) are metabolites of cholesterol produced in the human body. Oxysterols are implicated in many physiological and pathological processes and are shown to regulate the immune system. Recently, we demonstrated that during virus infections, 25HC amplifies proinflammatory response by directly binding to αvβ3 and α5β1 integrins and by activating FAK-NFkB signaling pathway, resulting in the production of proinflammatory mediators such as TNFα and IL-6. Surprisingly, our combined in silico and in vitro studies revealed that 25HC binds to integrins at a novel binding site (site 2) that is distinct from the classical ‘RGD’ binding site, where many extracellular matrix proteins with Arg-Gly-Asp motif are known to bind. Integrins are ubiquitous and play an important role in cell adhesion, cell-cell interactions, cell signaling, and extracellular matrix assembly. Given the abundance of both integrins and endogenous oxysterols, we hypothesized that other oxysterols (other than 25HC) might also activate integrins in a similar fashion. In this study, using a combined computational and experimental approach, we show that 24(S)-hydroxycholesterol (24HC) binds to integrin αvβ3 with high-affinity and triggers proinflammatory response via integrin-FAK pathway. Methods Molecular docking and all-atom molecular dynamics (MD) simulations were used to obtain binding poses and to study the ligand stability and conformational changes, respectively. Induced fit docking of 24HC at site 2 of αVβ3 ectodomain (PDB ID 3IIJE) was performed using MOE software. All-atom MD simulations were performed with GROMACS v5.1.2 using CHARMM36 force field and TIP3P water model. MD simulations were run in triplicates each for 200 ns. The proinflammatory response was determined by the production of TNF using ELISA. Results Our studies reveal the preferred binding orientation of 24HC, which is distinct from that of 25HC. Binding of 24HC induced structural changes in the specificity determining loop (SDL) of the βI domain, leading to an increase in solvent-accessible surface area (SASA) of the RGD site, disruption of native SDL-βI and SDL-β-propeller interface contacts, possibly altering the conformation of the RGD binding site. Robust production of TNF analyzed by ELISA indicates activation of the inflammatory response by 24HC. Similar to 25HC, 24HC also requires integrin-FAK pathway for inflammatory response since, FAK inhibitor, PF-431396 abrogated 24HC mediated response. Conclusions: As the primary cholesterol metabolite with exclusive production in the brain, these findings may provide key insight into factors that influence the complexities of brain function. Our study, for the first time, offers useful insight into the structural and mechanistic bases for the potential proinflammatory response mediated by 24HC through integrins and paves the way for drug discovery efforts towards identifying therapeutics in the treatment of infectious, inflammatory, and immune disorders.

Cryo-Electron Microscopy Structure of the αIIbβ3-Abciximab Complex.

The αIIbβ3 antagonist antiplatelet drug abciximab is the chimeric antigen-binding fragment comprising the variable regions of murine monoclonal antibody 7E3 and the constant domains of human IgG1 and light chain κ. Previous mutagenesis studies suggested that abciximab binds to the β3 C177-C184 specificity-determining loop (SDL) and Trp129 on the adjacent β1-α1 helix. These studies could not, however, assess whether 7E3 or abciximab prevents fibrinogen binding by steric interference, disruption of either the αIIbβ3-binding pocket for fibrinogen or the β3 SDL (which is not part of the binding pocket but affects fibrinogen binding), or some combination of these effects. To address this gap, we used cryo-electron microscopy to determine the structure of the αIIbβ3-abciximab complex at 2.8 Å resolution. Approach and Results: The interacting surface of abciximab is comprised of residues from all 3 complementarity-determining regions of both the light and heavy chains, with high representation of aromatic residues. Binding is primarily to the β3 SDL and neighboring residues, the β1-α1 helix, and β3 residues Ser211, Val212 and Met335. Unexpectedly, the structure also indicated several interactions with αIIb. As judged by the cryo-electron microscopy model, molecular-dynamics simulations, and mutagenesis, the binding of abciximab does not appear to rely on the interaction with the αIIb residues and does not result in disruption of the fibrinogen-binding pocket; it does, however, compress and reduce the flexibility of the SDL. We deduce that abciximab prevents ligand binding by steric interference, with a potential contribution via displacement of the SDL and limitation of the flexibility of the SDL residues.

Cryo-Electron Microscopy Structure of the aIIbβ3-Abciximab Complex

DN, YZ, AS, & JL contributed equally, as did MF, TW, & BSC The αIIbβ3 antagonist antiplatelet drug abciximab, approved in 1994, is the chimeric antigen-binding fragment (Fab) comprising the variable regions of murine mAb 7E3 and human IgG1 and light chain κ constant domains. In studies involving thousands of patients undergoing percutaneous coronary interventions, abciximab decreased mortality and the risk of recurrent myocardial infarction. Mutagenesis studies conducted by us and others (Puzon-McLaughlin, JBC 2000; Takagi, Biochem 2002; Artoni, PNAS 2004) suggested that abciximab binds to the β3 C177-C184 specificity-determining loop (SDL) and Trp129 on the adjacent β1-α1 helix, and our negative-stain electron microscopy (EM) studies of the complex of mAb 7E3 with αIIbβ3 in nanodiscs (Choi, Blood 2013) supported its binding to the αIIbβ3 head domain. None of these studies, however, had the resolution to assess whether 7E3 or abciximab prevents fibrinogen binding by steric interference, disruption of the αIIbβ3-binding pocket for fibrinogen, or both. To address this knowledge gap, we used cryo-EM to produce a density map at 2.8-Å resolution, which allowed us to build an atomic model of the αIIbβ3-abciximab complex. The interacting surface of abciximab is comprised of residues from all three complementarity determining regions of both the light and heavy chains, with high representation of aromatic residues (Figure). Abciximab buries a total of 1,273 Å2 of solvent-exposed surface on αIIbβ3, of which 1,040 Å2 is with β3 and 218 Å2 is with αIIb. The binding of abciximab does not result in disruption of the ADMIDAS, MIDAS, or SyMBS metal ion regions, but it does produce an ~3.4 Å compression of the SDL. Binding is primarily to the β3 SDL and neighboring residues, the β1-α1 helix, and β3 Ser211-Val212 and Met335. The latter residue interacts with the ADMIDAS metal ion in the unliganded receptor; ligand binding leads to the loss of the interaction and a dramatic swing-out motion of the β3 subunit that produces a high-affinity ligand-binding conformation. Surprisingly, the structure also indicated several abciximab interactions with αIIb. To assess the contribution of individual interactions between αIIbβ3 integrin and abciximab to the stability of the αIIbβ3-abciximab complex, we carried out 4 independent molecular dynamics simulations of the cryo-EM structure as well as the X-ray crystal structure of ligand-free αIIbβ3 integrin in its closed conformation (PDB: 3FCS). These revealed that the dynamic behavior of the RGD peptide-binding pocket was similar between the cryo-EM structure of the αIIbβ3-abciximab complex and the X-ray structure of unbound αIIbβ3 during the total simulation time of 2 microseconds per system. Abciximab-protein interaction analysis of the simulations demonstrated that: 1. The αIIb subunit participated marginally in the interaction with abciximab, with only the Asp159(αIIb)-Arg7(light chain) making contact for >0.5 fraction of the simulation time. 2. The β3 residues with >0.7 contact fraction values were Lys125, Asp126, Trp129, and Gln132 on the β1-α1 helix; Glu171, Asn175, Tyr178, Lys181, Thr182, and Thr183 on the SDL; and Val212, Met335, and Asp336. We calculated the effect of every possible mutation at each residue involved in significant inter-molecular interaction with abciximab in terms of changes in free energy of binding, and the resulting relative values were compared to experimental mutagenesis data. Thus, we made αIIb Asp159Ala and β3 Met335Asp mutations, the latter producing the analogous murine residue and the mutation predicted to be most disruptive to abciximab binding. We found no effect of either mutation on the binding of either mAb 7E3 or abciximab as judged by flow cytometry. Our data demonstrate unexpected interactions of abciximab with several αIIb residues and β3 Met335. Most importantly, abciximab binding did not alter the atomic structure or dynamics of the RGD-binding pocket in the timescale of the simulation, and so unless it induces allosteric modulation over a longer time scale, it does not appear to disrupt the RGD-binding pocket. Abciximab did, however, compress the SDL, which is not a component of the RGD-binding pocket but contributes to ligand binding by a still undefined mechanism. Thus, our data are most consistent with abciximab preventing ligand binding by steric interference, with a potential contribution via alteration of the SDL. Figure Disclosures Coller: Centocor/Janssen: Patents & Royalties: abxicimab; Accumetrics/Instrumentation Laboratory: Patents & Royalties: VerifyNow assay; Scholar Rock: Consultancy, Equity Ownership; CeleCor: Consultancy, Equity Ownership, Research Funding.

Cation-pi interaction regulates ligand-binding affinity and signaling of integrin alpha4beta7.

Integrin α(4)β(7) mediates rolling and firm adhesion of leucocytes, two of the critical steps in leukocyte migration and tissue specific homing. Affinity of α(4)β(7) for ligand is dynamically regulated by three interlinked metal ion-binding sites in β(7)-subunit I domain. In this study, we found that Phe185 (F185), a highly conserved aromatic residue in β(7)-subunit, links the specificity-determining loop and the synergistic metal ion-binding site (SyMBS) through cation-π interaction. Mutations of F185 that disrupted the SyMBS cation-F185 interaction led to deficient firm cell adhesion mediated by high affinity α(4)β(7), and only slightly affected rolling adhesion mediated by low affinity α(4)β(7). Disruption of SyMBS cation-F185 interaction induced partial extension of integrin ectodomain and separation of cytoplasmic tails, and impaired α(4)β(7)-mediated bidirectional signaling. In addition, loss of SyMBS cation-F185 interaction increased paxillin expression and promoted paxillin-integrin binding, leading to deficient cell spreading. Furthermore, integrin α(4)β(7)-mediated cell migration was decreased by the abolishment of SyMBS cation-F185 interaction. Thus, these findings reveal a cation-π interaction playing vital roles in the regulation of integrin affinity, signaling, and biological functions.

Targeted molecular dynamics reveals overall common conformational changes upon hybrid domain swing‐out in β3 integrins

The beta3 integrin family members alphaIIbeta3 and alphaVbeta3 signal bidirectionally through long-range allosteric changes, including a transition from a bent unliganded-closed low-affinity state to an extended liganded-open high-affinity state. To obtain an atomic-level description of this transition in an explicit solvent, we carried out targeted molecular dynamics simulations of the headpieces of alphaIIbeta3 and alphaVbeta3 integrins. Although minor differences were observed between these receptors, our results suggest a common transition pathway in which the hybrid domain swing-out is accompanied by conformational changes within the beta3 betaA (I-like) domain that propagate through the alpha7 helix C-terminus, and are followed by the alpha7 helix downward motion and the opening of the beta6-alpha7 loop. Breaking of contact interactions between the beta6-alpha7 loop and the alpha1 helix N-terminus results in helix straightening, internal rearrangements of the specificity determining loop (SDL), movement of the beta1-alpha1 loop toward the metal ion dependent adhesion site (MIDAS), and final changes at the interfaces between the beta3 betaA (I-like) domain and either the hybrid or the alpha beta-propeller domains. Taken together, our results suggest novel testable hypotheses of intradomain and interdomain interactions responsible for beta3 integrin activation.

Effect of altered glycosylation on the structure of the I‐like domain of β1 integrin: A molecular dynamics study

Glycosylation plays an important role in the regulation of integrin function. Molecular mechanisms underlying the effects of altered glycosylation on beta1 integrin structure and function are still largely unknown. In this study, we used a molecular modeling approach to study the effects of altered glycosylation, with alpha2-6 sialic acid and without alpha2-6 sialic acid, on the structure of the I-like domain of the beta1 integrin. Our results demonstrated that altered glycosylation affected the interactions between oligosaccharides and the I-like domain, which in turn changed the accessibility of the specificity-determining loop for ligand binding. Altered glycosylation caused significant conformational changes for most of the key functional regions of the I-like domain of beta1 integrin, including the metal ion-dependent adhesion site that contains a DLSYS motif, and other critical residues for ligand binding (Asn-224, Glu-229, Asp-233, Asp-267, and Asp-295). In addition, altered glycosylation caused significant movement of the alpha1 and alpha7 helices, which are important for the activation of beta1 integrin. The results from this study offered molecular mechanisms for the experimental observations that variant glycosylation regulates integrin function.

Three novel β3 domain-deletion peptides for the sensitive and specific detection of HPA-4 and six low frequency β3-HPA antibodies

Antibodies against human platelet antigens (HPA) are clinically important in fetal-maternal alloimmune thrombocytopenia, refractoriness to platelet transfusions and post-transfusion purpura. Of the 16 HPAs, nine are located on the beta3 subunit of the alphaIIb beta3 integrin. Antibody detection is generally based on platelet-derived alphaIIb beta3 from HPA-genotyped donors. Recombinant allelic beta3 peptides, expressed at high levels would improve consistency in antibody detection, but the expression of soluble and monomeric integrins expressing complex dependent epitopes has previously proved challenging. We aimed to generate three recombinant beta3 peptides for the detection of antibodies against HPA-4, HPA-8bw and five of the six remaining low frequency beta3 alloantigens. The removal of the specificity-determining loop from the betaA domain and fusion of truncated beta3 to calmodulin was exploited to obtain expression of monomeric protein. Using site-directed mutagenesis, the mutations for HPA-4b and HPA-8bw were introduced in the ITGB3*001 haplotype. A third peptide for the detection of antibodies against HPA coded by non-synonymous single nucleotide polymorphisms of low frequency was generated by the introduction of five mutations forming the basis of HPA-6bw, -7bw, -10bw, -11bw, and -16bw antigens. Reactivity of the three peptides with beta3-specific murine monoclonal antibodies and human HPA-1a phage antibodies confirmed the structural integrity of the recombinant fragments, and reactivity with a unique panel of polyclonal anti-HPA sera confirmed expression of the relevant HPA epitopes. These data demonstrate that beta3 integrin domain-deletion fragments are suitable molecular targets for HPA antibody detection.

Three novel beta3 domain-deletion peptides for the sensitive and specific detection of HPA-4 and six low frequency beta3-HPA antibodies.

Antibodies against human platelet antigens (HPA) are clinically important in fetal-maternal alloimmune thrombocytopenia, refractoriness to platelet transfusions and post-transfusion purpura. Of the 16 HPAs, nine are located on the beta3 subunit of the alphaIIb beta3 integrin. Antibody detection is generally based on platelet-derived alphaIIb beta3 from HPA-genotyped donors. Recombinant allelic beta3 peptides, expressed at high levels would improve consistency in antibody detection, but the expression of soluble and monomeric integrins expressing complex dependent epitopes has previously proved challenging. We aimed to generate three recombinant beta3 peptides for the detection of antibodies against HPA-4, HPA-8bw and five of the six remaining low frequency beta3 alloantigens. The removal of the specificity-determining loop from the betaA domain and fusion of truncated beta3 to calmodulin was exploited to obtain expression of monomeric protein. Using site-directed mutagenesis, the mutations for HPA-4b and HPA-8bw were introduced in the ITGB3*001 haplotype. A third peptide for the detection of antibodies against HPA coded by non-synonymous single nucleotide polymorphisms of low frequency was generated by the introduction of five mutations forming the basis of HPA-6bw, -7bw, -10bw, -11bw, and -16bw antigens. Reactivity of the three peptides with beta3-specific murine monoclonal antibodies and human HPA-1a phage antibodies confirmed the structural integrity of the recombinant fragments, and reactivity with a unique panel of polyclonal anti-HPA sera confirmed expression of the relevant HPA epitopes. These data demonstrate that beta3 integrin domain-deletion fragments are suitable molecular targets for HPA antibody detection.

Mutation of a Conserved Asparagine in the I-like Domain Promotes Constitutively Active Integrins αLβ2 and αIIbβ3

The leukocyte beta2 integrins are heterodimeric adhesion receptors required for a functional immune system. Many leukocyte adhesion deficiency-1 (LAD-1) mutations disrupt the expression and function of beta2 integrins. Herein, we further characterized the LAD-1 mutation N329S in the beta2 inserted (I)-like domain. This mutation converted alphaLbeta2 from a resting into a high affinity conformer because alphaLbeta2N329S transfectants adhered avidly to ligand intercellular adhesion molecule (ICAM)-3 in the absence of additional activating agent. An extended open conformation is adopted by alphaLbeta2N329S because of its reactivity with the beta2 activation reporter monoclonal antibodies MEM148 and KIM127. A corresponding mutation in beta3 generated constitutively active alphaIIbbeta3 that adhered to fibrinogen. This Asn is conserved in all human beta subunits, and it resides before the last helix of the I-like domain, which is known to be important in activation signal propagation. By mutagenesis studies and review of existing integrin structures, we conjectured that this conserved Asn may have a primary role in shaping the I-like domain by stabilizing the conformation of the alpha7 helix and the beta6-alpha7 loop in the I-like domain.

Equilibrium and Non-Equilibrium Molecular Dynamics Simulations Provide Potential Mechanisms for the Loss of Ligand Binding to αIIbβ3 Mutants Affecting the Ligand-Induced Metal Binding Site (LIMBS).

Although the role of the β3 MIDAS metal ion in ligand binding to αIIbβ3 is well established, serving as the site of interaction of the ligand Asp residue, the role of the nearby LIMBS metal ion is less well defined. Previous studies suggested a role for the LIMBS in ligand binding. We confirmed this by showing that HEK293 cells expressing normal αIIbβ3 adhered to both immobilized fibrinogen and the RGD-containing venom echistatin in the presence of either Mg++/Ca++ or Mn++, whereas two different αIIbβ3 LIMBS mutants (β3 N215A and D217A) failed to adhere to either protein. In addition, we found that both mutations also increased the binding of mAb AP5, which recognizes a ligand-induced binding site (LIBS) in the β3 PSI domain (normal 7±4% vs N215A 46±12% and D217A 41±20% of mAb anti-αIIb (HIP8) binding; mean±SD, n=6, p<0.05 for both), indicating that the mutations caused allosteric changes in the conformation of the receptor. To define the mechanism(s) by which the LIMBS mutants affect ligand binding, we carried out equilibrium and non-equilibrium (steered) molecular dynamics (MD) simulations of the cyclic peptide ligand eptifibatide in complex with either the fully hydrated normal αIIbβ3 integrin headpiece (PDB 1TY6) or the equivalent β3 D217A mutant, with and without the LIMBS metal ion. Simulations were carried out using the GROMACS package with the OPLS all-atom force-field. During the simulation, the hybrid domain of the D217A mutant demonstrated greater structural fluctuations than the normal αIIbβ3. Although Craig et al. have reported the appearance of a new contact between the RGD peptide ligand Asp carboxyl and the LIMBS metal ion in αVβ3 after 10 ps of a 1 ns simulation, we did not observe the appearance of such an interaction between the eptifibatide carboxyl and the normal αIIbβ3 LIMBS metal ion even after 20 ns. We did, however, observe such an interaction with the LIMBS metal ion in the D217A mutant. This interaction was facilitated by the movement of the LIMBS ~ 2 Å closer to the MIDAS, and was accompanied by rearrangements of the LIMBS coordinating residues D158 and N215. When the D217A mutant simulation was performed in the absence of the LIMBS metal ion, changes in the orientation of E220 were also observed. The D217A mutant demonstrated increased fluctuations in the C177–C184 specificity-determining loop (SDL), which has been implicated in ligand binding, and decreased fluctuations in K209. Steered MD were used to investigate the pulling forces required to unbind eptifibatide from its binding site. Notably, although the unbinding force decreased modestly when the LIMBS metal ion was removed, it required removal of both the LIMBS and MIDAS metal ions to effect a marked reduction in unbinding force. The binding free energies of the association of the αIIb and β3 subunits were also calculated, and the D217A mutant in the presence of the LIMBS metal ion demonstrated much tighter binding than normal integrin αIIbβ3 (ΔGb −162±6 vs −119±6 Kcal/mol; mean±SD; n=500). We conclude that the LIMBS plays a crucial role in ligand binding to αIIbβ3, perhaps by virtue of its effects on the coordination of the MIDAS, the accentuated mobility of specific domains (e.g., the SDL and the hybrid domains), and/or the number and strength of contacts between αIIb and β3.

Neither Free αIIb nor Free β3 Limits αIIbβ3 Biogenesis, and Pro-αIIbβ3 Adopts a Conformation Akin to Ligand-Bound Mature αIIbβ3.

The first steps in the biogenesis of αIIbβ3 involve the individual synthesis of each subunit within the endoplasmic reticulum, followed by complex formation. Previous studies performed in HEL cells and in megakaryocyte-lineage cells cultured from cryopreserved progenitor cells of patients with CML have led to conflicting data with regard to whether αIIb or β3 is produced in limiting amounts that might then control complex formation. To study this question in greater detail, we conducted immunoprecipitation studies on HEK293 cells transfected with αIIb and β3 cDNAs and megakaryocyte-lineage cells derived from human umbilical cord blood grown in the presence of thrombopoietin and IL-11. Cells were labeled for 15 min with 35S-methionine/cysteine, and then lysed after 2 hr with a 1% triton X-100 buffer. The antibodies employed included murine monoclonal antibodies 10E5 (anti-αIIbβ3 complex, which, reacts with αIIb cap domain), 7E3 (anti-αIIbβ3 + αvβ3, which reacts with β3 specificity-determining loop and α1 helix), AP-5 (LIBS anti-β3, which reacts with β3 amino acids 1 - 6), 7H2 (anti-β3, which reacts with PSI domain near C13), 1990 (anti-αIIb, which reacts at unknown site), and LIBS2 (LIBS anti-β3, which reacts with a site within amino acids 602 – 690). The relative amounts of pro-αIIb, mature αIIb, and β3 immunoprecipitated by this panel of antibodies is shown in Table 1. Results were similar in both the HEK293 cells and in megakaryocyte-lineage cells derived from human umbilical cord blood. These data indicate that there are sizeable pools of both free pro-αIIb and free β3 in both cell types, and thus neither subunit controls complex formation by limited availability. Based on the selective precipitation of pro-αIIb + β3 by AP5, it appears that the pro-αIIbβ3 complex adopts a conformation similar to that of ligand-bound mature αIIbβ3. We conclude that αIIbβ3 complex formation is slow relative to the production of the individual subunits, and is probably controlled either by the ability of the subunits to adopt the proper fold and/or to interact with a chaperone(s) that facilitates complex formation. Assuming that AP5 selectively recognizes αIIbβ3 complexes in which the angle of the β3 βA (I-like) domain - hybrid domain interface results in separation of the αIIb and β3 leg domains, these data also suggest that αIIbβ3 head-head interactions precede αIIbβ3 leg-leg interactions during biogenesis, and that αIIb maturation occurs rapidly after αIIbβ3 leg-leg interactions. Such a model is consistent with our previous data demonstrating that pro-αIIb binds to the ER membrane-resident chaperone calnexin via the N-linked glycan at αIIb N15.Table 1AntibodyPro- αIIbMature αIIbβ31990++++++++++10E5+++++++7E3+++++++7H2+++++++++LIBS2+−++++AP5++−++++

Crucial Role of the Specificity-determining Loop of the Integrin β4 Subunit in the Binding of Cells to Laminin-5 and Outside-in Signal Transduction

Within each hemidesmosome, alpha6beta4 integrin plays a crucial role in hemidesmosome assembly by binding to laminin-5 in the basement membrane zone of epithelial tissue. Recent analyses have implicated "specificity-determining loops" (SDLs) in the I-like domain of beta integrin in regulating ligand binding. Here, we investigated the function of an SDL-like motif within the extracellular I-like domain of beta4 integrin. We generated point mutations within the SDL of beta4 integrin tagged with green fluorescent protein (GFP-beta4K150A and GFP-beta4Q155L). We also generated a mutation within the I-like domain of the beta4 integrin, lying outside the SDL region (GFP-beta4V284E). We transfected constructs encoding the mutated beta4 integrins and a GFP-conjugated wild type beta4 integrin (GFP-beta4WT) into 804G cells, which assemble hemidesmosomes, and human endothelial cells, which express little endogenous beta4 integrin. In transfected 804G cells, GFP-beta4WT and GFP-beta4V284E colocalize with hemidesmosome proteins, whereas hemidesmosomal components in cells expressing GFP-beta4K150A and GFP-beta4Q155L are aberrantly localized. In endothelial cells, GFP-beta4WT and mutant proteins are co-expressed at the cell surface with alpha6 integrin. When transfected endothelial cells are plated onto laminin-5 matrix, GFP-beta4WT and GFP-beta4V284E localize with laminin-5, whereas GFP-beta4K150A and GFP-beta4Q155L do not. GFP-beta4WT and GFP-beta4V284E expressed in endothelial cells associate with the adaptor protein Shc when the cells are stimulated with laminin-5. However, GFP-beta4K150A and GFP-beta4Q155L fail to associate with Shc even when laminin-5 is present, thus impacting downstream signaling. These results provide evidence that the SDL segment of the beta4 integrin subunit is required for ligand binding and is involved in outside-in signaling.

The role of the specificity-determining loop of the integrin beta subunit I-like domain in autonomous expression, association with the alpha subunit, and ligand binding.

Integrin beta subunits contain a highly conserved I-like domain that is known to be important for ligand binding. Unlike integrin I domains, the I-like domain requires integrin alpha and beta subunit association for optimal folding. Pactolus is a novel gene product that is highly homologous to integrin beta subunits but lacks associating alpha subunits [Chen, Y., Garrison, S., Weis, J. J., and Weis, J. H. (1998) J. Biol. Chem. 273, 8711-8718] and a approximately 30 amino acid segment corresponding to the specificity-determining loop (SDL) in the I-like domain. We find that the SDL is responsible for the defects in integrin beta subunit expression and folding in the absence of alpha subunits. When transfected in the absence of alpha subunits into cells, extracellular domains of mutant beta subunits lacking SDL, but not wild-type beta subunits, were well secreted and contained immunoreactive I-like domains. The purified recombinant soluble beta1 subunit with the SDL deletion showed an elongated shape in electron microscopy, consistent with its structure in alphabeta complexes. The SDL segment is not required for formation of alpha5beta1, alpha4beta1, alphaVbeta3, and alpha6beta4 heterodimers, but is essential for fomation of alpha6beta1, alphaVbeta1, and alphaLbeta2 heterodimers, suggesting that usage of subunit interface residues is variable among integrins. The beta1 SDL is required for ligand binding and for the formation of the epitope for the alpha5 monoclonal antibody 16 that maps to loop segments connecting blades 2 and 3 of beta-propeller domain of alpha5, but is not essential for nearby beta-propeller epitopes.

Structural and Functional Studies with Antibodies to the Integrin β2 Subunit: A MODEL FOR THE I-LIKE DOMAIN

To establish a structure and function map of the beta2 integrin subunit, we mapped the epitopes of a panel of beta2 monoclonal antibodies including function-blocking, nonblocking, and activating antibodies using human/mouse beta2 subunit chimeras. Activating antibodies recognize the C-terminal half of the cysteine-rich region, residues 522-612. Antibodies that do not affect ligand binding map to residues 1-98 and residues 344-521. Monoclonal antibodies to epitopes within a predicted I-like domain (residues 104-341) strongly inhibit LFA-1-dependent adhesion. These function-blocking monoclonal antibodies were mapped to specific residues with human --> mouse knock-out or mouse --> human knock-in mutations. Combinatorial epitopes involving residues distant in the sequence provide support for a specific alignment between the beta-subunit and I domains that was used to construct a three-dimensional model. Antigenic residues 133, 332, and 339 are on the first and last predicted alpha-helices of the I-like domain, which are adjacent on its "front." Other antigenic residues in beta2 and in other integrin beta subunits are present on the front. No antigenic residues are present on the "back" of the domain, which is predicted to be in an interface with other domains, such as the alpha subunit beta-propeller domain. Most mutations in the beta2 subunit in leukocyte adhesion deficiency are predicted to be buried in the beta2 subunit I-like domain. Two long insertions are present relative to alpha-subunit I-domains. One is tied down to the back of the I-like domain by a disulfide bond. The other corresponds to the "specificity-determining loop" defined in beta1 and beta3 integrins and contains the antigenic residue Glu(175) in a disulfide-bonded loop located near the "top" of the domain.

The Structural Features of Concanavalin A Governing Non-proline Peptide Isomerization

The reversible binding of manganese and calcium to concanavalin A determines the carbohydrate binding of the lectin by inducing large conformational changes. These changes are governed by the isomerization of a non-proline peptide bond, Ala-207-Asp-208, positioned in a beta-strand in between the calcium binding site S2 and the carbohydrate specificity-determining loop. The replacement of calcium by manganese allowed us to investigate the structures of the carbohydrate binding, locked state and the inactive, unlocked state of concanavalin A, both with and without metal ions bound. Crystals of unlocked metal-free concanavalin A convert to the locked form with the binding of two Mn(2+) ions. Removal of these ions from the crystals traps metal-free concanavalin A in its locked state, a minority species in solution. The ligation of a metal ion in S2 to unlocked concanavalin A causes bending of the beta-strand foregoing the S2 ligand residues Asp-10 and Tyr-12. This bending disrupts conventional beta-sheet hydrogen bonding and forces the Thr-11 side chain against the Ala-207-Asp-208 peptide bond. The steric strain exerted by Thr-11 is presumed to drive the trans-to-cis isomerization. Upon isomerization, Asp-208 flips into its carbohydrate binding position, and the conformation of the carbohydrate specificity determining loop changes dramatically.