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Bing-Hao Luo

Publications and source records attributed to Bing-Hao Luo.

12 recordsLinked to original sources
PubMed2007

Structural basis of integrin regulation and signaling.

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.

Animals↗
PubMed2006

Integrin structures and conformational signaling.

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.

Animals↗
PubMed2005

Conformational studies of the tetramerization site of human erythroid spectrin by cysteine-scanning spin-labeling EPR methods.

We used cysteine-scanning and spin-labeling methods to prepare singly spin labeled recombinant peptides for electron paramagnetic resonance studies of the partial domain regions at the tetramerization site (N-terminal end of alpha and C-terminal end of beta) of erythroid spectrin. The values of the inverse line width parameter (deltaH0(-1)) from a family of Sp alphaI-1-368delta peptides scanning residues 21-30 exhibited a periodicity of approximately 3.5-4. We used molecular dynamics calculations to show that the asymmetric mobility of this helix is not necessarily due to tertiary contacts, but is likely due to intrinsic properties of helix C', a helix with a heptad pattern sequence. The residues with low deltaH0(-1) values (residues at positions 21, 25, and 28/29) were those on the hydrophobic side of this amphipathic helix. Native gel electrophoresis results showed that these residues were functionally important and are involved in the tetramerization process. Thus, EPR results readily identified functionally important residues in the alpha spectrin partial domain region. Mutations at these positions may lead to clinical symptoms. Similarly, the deltaH0(-1) values from a family of spin-labeled Sp betaI-1898-2083delta peptides also exhibited a periodicity of approximately 3.5-4, indicating a helical conformation in the two scanned regions (residues 2008-2018 and residues 2060-2070). However, the region consisting of residues 2071-2076 was in a disordered conformation. Both helical regions include a hydrophilic side with high deltaH0(-1) values and a hydrophobic side with low deltaH0(-1) values, demonstrating the amphipathic nature of the helical regions. Residues 2008, 2011, 2014, and 2018 in the first scanned region and residues 2061, 2065, and 2068 in the second scanned region were on the hydrophobic side. These residues were critical in alphabeta spectrin association at the tetramerization site. Mutations at some of these positions have been reported to be detrimental in clinical studies.

Cyclic N-Oxides↗
PubMed2005

Disrupting integrin transmembrane domain heterodimerization increases ligand binding affinity, not valency or clustering.

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.

Amino Acid Motifs↗
PubMed2004

The relative influence of metal ion binding sites in the I-like domain and the interface with the hybrid domain on rolling and firm adhesion by integrin alpha4beta7.

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.

Amino Acid Sequence↗
PubMed2004

A specific interface between integrin transmembrane helices and affinity for ligand.

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.

Amino Acid Sequence↗
PubMed2004

Allosteric beta1 integrin antibodies that stabilize the low affinity state by preventing the swing-out of the hybrid domain.

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.

Allosteric Regulation↗
PubMed2003

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.

Animals↗
PubMed2003

Stabilizing the open conformation of the integrin headpiece with a glycan wedge increases affinity for ligand.

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.

Animals↗
PubMed2003

High affinity ligand binding by integrins does not involve head separation.

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.

Animals↗
PubMed2002

Important region in the beta-spectrin C-terminus for spectrin tetramer formation.

Many hereditary hemolytic anemias are due to spectrin mutations at the C-terminal region of beta-spectrin (the betaC region) that destabilize spectrin tetramer formation. However, little is known about the betaC region of spectrin. We have prepared four recombinant beta-peptides of different lengths from human erythrocyte spectrin, all starting at position 1898 of the C-terminal region, but terminating at position 2070, 2071, 2072 or 2073. Native polyacrylamide gel electrophoresis showed that the two peptides terminating at positions 2070 and 2071 did not associate with an N-terminal region alpha-peptide (Spalpha1-156) in the micromolar range. However, the peptides that terminated at positions 2072 and 2073 associated with the alpha-peptide. Circular dichroism results showed that the unassociated helices in both alpha- and beta-peptides became associated, presumably to form a helical bundle, for those beta-peptides that formed an alphabeta complex, but not for those beta-peptides that did not form an alphabeta complex. In addition, upon association, an increase in the alpha-helical content was observed. These results showed that the beta-peptides ending prior to residue 2072 (Thr) would not associate with alpha-peptide, and that no helical bundling of the partial domains was observed. Thus, we suggest that the C-terminal segment of beta-spectrin, starting from residue 2073 (Thr), is not critical to spectrin tetramer formation. However, the C-terminal region ending with residue 2072 is important for its association with alpha-spectrin in forming tetramers.

Amino Acid Sequence↗