Bing-Hao Luo

Louisiana State University, Baton Rouge, LA, United States

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Publications (18)161.62 Total impact

  • [show abstract] [hide abstract]
    ABSTRACT: Many questions about the significance of structural features of integrin α(V)β(3) with respect to its mechanism of activation remain. We have determined and re-refined crystal structures of the α(V)β(3) ectodomain linked to C-terminal coiled coils (α(V)β(3)-AB) and four transmembrane (TM) residues in each subunit (α(V)β(3)-1TM), respectively. The α(V) and β(3) subunits with four and eight extracellular domains, respectively, are bent at knees between the integrin headpiece and lower legs, and the headpiece has the closed, low-affinity conformation. The structures differ in the occupancy of three metal-binding sites in the βI domain. Occupancy appears to be related to the pH of crystallization, rather than to the physiologic regulation of ligand binding at the central, metal ion-dependent adhesion site. No electron density was observed for TM residues and much of the α(V) linker. α(V)β(3)-AB and α(V)β(3)-1TM demonstrate flexibility in the linker between their extracellular and TM domains, rather than the previously proposed rigid linkage. A previously postulated interface between the α(V) and β(3) subunits at their knees was also not supported, because it lacks high-quality density, required rebuilding in α(V)β(3)-1TM, and differed markedly between α(V)β(3)-1TM and α(V)β(3)-AB. Together with the variation in domain-domain orientation within their bent ectodomains between α(V)β(3)-AB and α(V)β(3)-1TM, these findings are compatible with the requirement for large structural changes, such as extension at the knees and headpiece opening, in conveying activation signals between the extracellular ligand-binding site and the cytoplasm.
    Biochemistry 10/2012; · 3.38 Impact Factor
  • Ping Hu, Bing-Hao Luo
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    ABSTRACT: Integrins are heterodimeric cell adhesion molecules that are important in many biological functions such as cell migration, proliferation and differentiation and survival. They can transmit bidirectional signals across the plasma membrane. Inside-out activating signal from some cell surface receptors bound with soluble agonists triggers integrins conformational change leading to high affinity for extracellular ligands. Then binding of ligands to integrins results in outside-in signaling, leading to formation of focal adhesion complex at the integrin cytoplasmic tail and activation of downstream signal pathways. This bi-directional signaling is essential for rapid response of cell to surrounding environmental changes. During this process, the conformational change of integrin extracellular and transmembrane/cytoplasmic domains is particularly important. In this review, we will summarize recent progress in both inside-out and outside-in signaling with specific focus on the mechanism how integrins transmit bidirectional signals through transmembrane/cytoplasmic domains. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.
    Journal of Cellular Physiology 07/2012; · 4.22 Impact Factor
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    ABSTRACT: Integrin transmembrane (TM) and/or cytoplasmic domains play a critical role in integrin bidirectional signaling. Although it has been shown that TM and/or cytoplasmic α and β domains associate in the resting state and separation of these domains is required for both inside-out and outside-in signaling, the role of TM homomeric association remains elusive. Formation of TM homo-oligomers was observed in micelles and bacterial membranes previously, and it has been proposed that homomeric association is important for integrin activation and clustering. This study addresses whether integrin TM domains form homo-oligomers in mammalian cell membranes using cysteine scanning mutagenesis. Our results show that TM homomeric interaction does not occur before or after soluble ligand binding or during inside-out activation. In addition, even though the cysteine mutants and the heterodimeric disulfide-bounded mutant could form clusters after adhering to immobilized ligand, the integrin TM domains do not form homo-oligomers, suggesting that integrin TM homomeric association is not critical for integrin clustering or outside-in signaling. Therefore, integrin TM homo-oligomerization is not required for integrin activation, ligand binding, or signaling.
    Journal of Biological Chemistry 11/2010; 286(3):1860-7. · 4.65 Impact Factor
  • Wei Wang, Bing-Hao Luo
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    ABSTRACT: Integrins are cell adhesion receptors that transmit bidirectional signals across plasma membrane and are crucial for many biological functions. Recent structural studies of integrin transmembrane (TM) and cytoplasmic domains have shed light on their conformational changes during integrin activation. A structure of the resting state was solved based on Rosetta computational modeling and experimental data using intact integrins on mammalian cell surface. In this structure, the alpha(IIb) GXXXG motif and their beta(3) counterparts of the TM domains associate with ridge-in-groove packing, and the alpha(IIb) GFFKR motif and the beta(3) Lys-716 in the cytoplasmic segments play a critical role in the alpha/beta association. Comparing this structure with the NMR structures of the monomeric alpha(IIb) and beta(3) (represented as active conformations), the alpha subunit helix remains similar after dissociation whereas beta subunit helix is tilted by embedding additional 5-6 residues into the lipid bilayer. These conformational changes are critical for integrin activation and signaling across the plasma membrane. We thus propose a new model of integrin TM activation in which the recent NMR structure of the alpha(IIb)beta(3) TM/cytoplasmic complex represents an intermediate or transient state, and the electrostatic interaction in the cytoplasmic region is important for priming the initial alpha/beta association, but not absolutely necessary for the resting state.
    Journal of Cellular Biochemistry 11/2009; 109(3):447-52. · 3.06 Impact Factor
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    ABSTRACT: Structures of intact receptors with single-pass transmembrane domains are essential to understand how extracellular and cytoplasmic domains regulate association and signaling through transmembrane domains. A chemical and computational method to determine structures of the membrane regions of such receptors on the cell surface is developed here and validated with glycophorin A. An integrin heterodimer structure reveals association over most of the lengths of the alpha and beta transmembrane domains and shows that the principles governing association of hetero and homo transmembrane dimers differ. A turn at the Gly of the juxtamembrane GFFKR motif caps the alpha TM helix and brings the two Phe of GFFKR into the alpha/beta interface. A juxtamembrane Lys residue in beta also has an important role in the interface. The structure shows how transmembrane association/dissociation regulates integrin signaling. A joint ectodomain and membrane structure shows that substantial flexibility between the extracellular and TM domains is compatible with TM signaling.
    Molecular cell 05/2009; 34(2):234-49. · 14.61 Impact Factor
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    ABSTRACT: The complete ectodomain of integrin alpha(IIb)beta(3) reveals a bent, closed, low-affinity conformation, the beta knee, and a mechanism for linking cytoskeleton attachment to high affinity for ligand. Ca and Mg ions in the recognition site, including the synergistic metal ion binding site (SyMBS), are loaded prior to ligand binding. Electrophilicity of the ligand-binding Mg ion is increased in the open conformation. The beta(3) knee passes between the beta(3)-PSI and alpha(IIb)-knob to bury the lower beta leg in a cleft, from which it is released for extension. Different integrin molecules in crystals and EM reveal breathing that appears on pathway to extension. Tensile force applied to the extended ligand-receptor complex stabilizes the closed, low-affinity conformation. By contrast, an additional lateral force applied to the beta subunit to mimic attachment to moving actin filaments stabilizes the open, high-affinity conformation. This mechanism propagates allostery over long distances and couples cytoskeleton attachment of integrins to their high-affinity state.
    Molecular cell 01/2009; 32(6):849-61. · 14.61 Impact Factor
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    ABSTRACT: Integrins are important cell surface receptors that transmit bidirectional signals across the membrane. It has been shown that a conformational change of the integrin beta-subunit headpiece (i.e. the beta I domain and the hybrid domain) plays a critical role in regulating integrin ligand binding affinity and function. Previous studies have used coarse methods (a glycan wedge, mutations in transmembrane contacts) to force the beta-subunit into either the open or closed conformation. Here, we demonstrate a detailed understanding of this conformational change by applying computational design techniques to select five amino acid side chains that play an important role in the energetic balance between the open and closed conformations of alphaIIbbeta3. Eight single-point mutants were designed at these sites, of which five bound ligands much better than wild type. Further, these mutants were found to be in a more extended conformation than wild type, suggesting that the conformational change at the ligand binding headpiece was propagated to the legs of the integrin. This detailed understanding of the conformational change will assist in the development of allosteric drugs that either stabilize or destabilize specific integrin conformations without occluding the ligand-binding site.
    Journal of Biological Chemistry 12/2008; 284(6):3917-24. · 4.65 Impact Factor
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    ABSTRACT: Adhesion to extracellular ligands through integrins regulates cell shape, migration, growth, and survival. How integrins transmit signals in the outside-to-in direction remains unknown. Whereas in resting integrins the alpha and beta subunit transmembrane domains are associated, ligand binding promotes dissociation and separation of these domains. Here we address whether such separation is required for outside-in signaling. By introduction of an intersubunit disulfide bond, we generated mutant integrin alphaIIbbeta3 with blocked transmembrane separation that binds ligand, mediates adhesion, adopts an extended conformation after ligand binding, and forms antibody-induced macroclusters on the cell surface similarly to wild type. However, the mutant integrin exhibits a profound defect in adhesion-induced outside-in signaling as measured by cell spreading, actin stress-fiber and focal adhesion formation, and focal adhesion kinase activation. This defect was rescued by reduction of the disulfide bond. Our results demonstrate that the separation of transmembrane domains is required for integrin outside-in signal transduction.
    Blood 11/2007; 110(7):2475-83. · 9.06 Impact Factor
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    ABSTRACT: 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.
    Journal of Biological Chemistry 05/2007; 282(16):11914-20. · 4.65 Impact Factor
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    ABSTRACT: Integrins are cell adhesion molecules that mediate cell-cell, cell-extracellular matrix, and cell-pathogen interactions. They play critical roles for the immune system in leukocyte trafficking and migration, immunological synapse formation, costimulation, and phagocytosis. Integrin adhesiveness can be dynamically regulated through a process termed inside-out signaling. In addition, ligand binding transduces signals from the extracellular domain to the cytoplasm in the classical outside-in direction. Recent structural, biochemical, and biophysical studies have greatly advanced our understanding of the mechanisms of integrin bidirectional signaling across the plasma membrane. Large-scale reorientations of the ectodomain of up to 200 A couple to conformational change in ligand-binding sites and are linked to changes in alpha and beta subunit transmembrane domain association. In this review, we focus on integrin structure as it relates to affinity modulation, ligand binding, outside-in signaling, and cell surface distribution dynamics.
    Annual Review of Immunology 02/2007; 25:619-47. · 36.56 Impact Factor
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    Bing-Hao Luo, Timothy A Springer
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    ABSTRACT: Integrins are cell adhesion molecules that play critical roles in development, wound healing, hemostasis, immunity and cancer. Advances in the past two years have shed light on the structural basis for integrin regulation and signaling, especially on how global conformational changes between bent and extended conformations relate to the inter-domain and intra-domain shape shifting that regulates affinity for ligand. The downward movements of the C-terminal helices of the alpha I and beta I domains and the swing-out of the hybrid domain play pivotal roles in integrin conformational signaling. Experiments have also shown that integrins transmit bidirectional signals across the plasma membrane by coupling extracellular conformational change with an unclasping and separation of the alpha and beta transmembrane and cytoplasmic domains.
    Current Opinion in Cell Biology 11/2006; 18(5):579-86. · 11.41 Impact Factor
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    ABSTRACT: Residues important in the interaction between the 23-residue transmembrane (TM) domains of the integrin alpha(IIb)- and beta(3)-subunits were identified by mutating each non-Leu residue to Leu. Leu substitutions of alpha(IIb) at G972, G976, and T981, and of beta(3) at I693 and G708, increased ligand binding. Substitutions with other amino acids at alpha(IIb)G972 and beta(3)G708 could also increase ligand binding. The results are consistent with and extend the helical interface between the integrin alpha- and beta-subunit TM domains previously defined by cysteine scanning and disulfide bond formation. We differentiated between affinity- and valency-based modes of activation by TM domain mutations. The mutant alpha(IIb) W967C forms disulfide-linked alpha(IIb)-subunits within an (alpha(IIb)beta(3))(2) tetramer. This tetramer behaved as an ideal model for the valency mode of regulation, because it exhibited significantly increased binding to multivalent but not monovalent ligands and basally retained the bent conformation. By contrast, the activating Leu mutants showed increased binding to the monovalent, ligand-mimetic PAC-1 Fab and increased exposure of ligand-induced binding site (LIBS) epitopes, suggesting that they partially adopt an extended conformation. Furthermore, the previously described beta(3)G708N mutation in Chinese hamster ovary cells enhanced ligand binding affinity, not valency, and did not alter cell-surface clustering as defined by confocal microscopy. Our studies provide evidence that disrupting the integrin heterodimeric TM helix-helix interface activates ligand binding mainly by increasing the monomeric affinity for ligand, but not the receptor valency, i.e., clustering.
    Proceedings of the National Academy of Sciences 04/2005; 102(10):3679-84. · 9.74 Impact Factor
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    ABSTRACT: We examined the effect of conformational change at the beta(7) I-like/hybrid domain interface on regulating the transition between rolling and firm adhesion by integrin alpha(4)beta(7). An N-glycosylation site was introduced into the I-like/hybrid domain interface to act as a wedge and to stabilize the open conformation of this interface and hence the open conformation of the alpha(4) beta(7) headpiece. Wild-type alpha(4)beta(7) mediates rolling adhesion in Ca(2+) and Ca(2+)/Mg(2+) but firm adhesion in Mg(2+) and Mn(2+). Stabilizing the open headpiece resulted in firm adhesion in all divalent cations. The interaction between metal binding sites in the I-like domain and the interface with the hybrid domain was examined in double mutants. Changes at these two sites can either counterbalance one another or be additive, emphasizing mutuality and the importance of multiple interfaces in integrin regulation. A double mutant with counterbalancing deactivating ligand-induced metal ion binding site (LIMBS) and activating wedge mutations could still be activated by Mn(2+), confirming the importance of the adjacent to metal ion-dependent adhesion site (ADMIDAS) in integrin activation by Mn(2+). Overall, the results demonstrate the importance of headpiece allostery in the conversion of rolling to firm adhesion.
    Journal of Biological Chemistry 01/2005; 279(53):55556-61. · 4.65 Impact Factor
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    ABSTRACT: The ligand binding function of integrins can be modulated by various monoclonal antibodies by both direct and indirect mechanisms. We have characterized an anti-beta(1) antibody, SG/19, that had been reported to inhibit the function of the beta(1) integrin on the cell surface. SG/19 recognized the wild type beta(1) subunit that exists in a conformational equilibrium between the high and low affinity states but bound poorly to a mutant beta(1) integrin that had been locked in a high affinity state. Epitope mapping of SG/19 revealed that Thr(82) in the beta(1) subunit, located at the outer face of the boundary between the I-like and hybrid domains, was the key binding determinant for this antibody. Direct visualization of the alpha (5)beta(1) headpiece fragment in complex with SG/19 Fab with electron microscopy confirmed the location of the binding surface and showed that the ligand binding site is not occluded by the bound Fab. Surface plasmon resonance showed that alpha (5)beta(1) integrin bound by SG/19 maintained a low affinity toward its physiological ligand fibronectin (Fn) whereas binding by function-blocking anti-alpha(5) antibodies resulted in a complete loss of fibronectin binding. Thus a class of the anti-beta antibodies represented by SG/19 attenuate the ligand binding function by restricting the conformational shift to the high affinity state involving the swing-out of the hybrid domain without directly interfering with ligand docking.
    Journal of Biological Chemistry 07/2004; 279(26):27466-71. · 4.65 Impact Factor
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    ABSTRACT: Conformational communication across the plasma membrane between the extracellular and intracellular domains of integrins is beginning to be defined by structural work on both domains. However, the role of the alpha and beta subunit transmembrane domains and the nature of signal transmission through these domains have been elusive. Disulfide bond scanning of the exofacial portions of the integrin alpha(IIbeta) and beta(3) transmembrane domains reveals a specific heterodimerization interface in the resting receptor. This interface is lost rather than rearranged upon activation of the receptor by cytoplasmic mutations of the alpha subunit that mimic physiologic inside-out activation, demonstrating a link between activation of the extracellular domain and lateral separation of transmembrane helices. Introduction of disulfide bridges to prevent or reverse separation abolishes the activating effect of cytoplasmic mutations, confirming transmembrane domain separation but not hinging or piston-like motions as the mechanism of transmembrane signaling by integrins.
    PLoS Biology 07/2004; 2(6):e153. · 12.69 Impact Factor
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    ABSTRACT: 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.
    Journal of Biological Chemistry 04/2004; 279(11):10215-21. · 4.65 Impact Factor
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    ABSTRACT: Conformational change in the integrin extracellular domain is required for high affinity ligand binding and is also involved in post-ligand binding cellular signaling. Although there is evidence to the contrary, electron microscopic studies showing that ligand binding triggers alpha- and beta-subunit dissociation in the integrin headpiece have gained popularity and support the hypothesis that head separation activates integrins. To test directly the head separation hypothesis, we enforced head association by introducing disulfide bonds across the interface between the alpha-subunit beta-propeller domain and the beta-subunit I-like domain. Basal and activation-dependent ligand binding by alpha(IIb)beta(3) and alpha(V)beta(3) was unaffected. The covalent linkage prevented dissociation of alpha(IIb)beta(3) into its subunits on EDTA-treated cells. Whereas EDTA dissociated wild type alpha(IIb)beta(3) on the cell surface, a ligand-mimetic Arg-Gly-Asp peptide did not, as judged by binding of complex-specific antibodies. Finally, a high affinity ligand-mimetic compound stabilized noncovalent association between alpha(IIb) and beta(3) headpiece fragments in the presence of SDS, indicating that ligand binding actually stabilized subunit association at the head, as opposed to the suggested subunit separation. The mechanisms of conformational regulation of integrin function should therefore be considered in the context of the associated alphabeta headpiece.
    Journal of Biological Chemistry 06/2003; 278(19):17185-9. · 4.65 Impact Factor
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    ABSTRACT: The affinity of the extracellular domain of integrins for ligand is regulated by conformational changes signaled from the cytoplasm. Alternative types of conformational movement in the ligand-binding headpiece have been proposed. In one study, electron micrograph image averages of the headpiece of integrin aV beta 3 show two different conformations. The open conformation of the headpiece is present when a ligand mimetic peptide is bound and differs from the closed conformation in the presence of an obtuse angle between the beta 3 subunit hybrid and I-like domains. We tested the hypothesis that opening of the hybrid-I-like domain interface increases ligand-binding affinity by mutationally introducing an N-glycosylation site into it. Both beta 3 and beta1 integrin glycan wedge mutants exhibit constitutively high affinity for physiological ligands. The data uniquely support one model of integrin activation and suggest that movement at the interface with the hybrid domain pulls down the C-terminal helix of the I-like domain and activates its metal ion-dependent adhesion site, analogously to activation of the integrin I domain.
    Proceedings of the National Academy of Sciences 04/2003; 100(5):2403-8. · 9.74 Impact Factor

Publication Stats

1k Citations
5 Downloads
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161.62 Total Impact Points

Institutions

  • 2009–2012
    • Louisiana State University
      • Department of Biological Sciences
      Baton Rouge, LA, United States
    • Boston Children's Hospital
      • Department of Pathology
      Boston, Massachusetts, United States
  • 2003–2009
    • Harvard Medical School
      • • Immune Disease Institute
      • • Department of Pathology
      Boston, MA, United States