Integrin α\u3csub\u3ev\u3c/sub\u3eβ\u3csub\u3e8\u3c/sub\u3e Adopts a High Affinity State for Soluble Ligands Under Physiological Conditions

© 2016 Wiley Periodicals, Inc. It has been proposed that integrins adopt a low affinity conformation under physiological conditions. Integrin can either be activated through cytoplasm or by binding of cations such as Mn2+ to the head domain. The cytoplasmic activation pathway, that is, inside-out signaling has been regarded as the physiological pathway for integrin activation. Integrin β8 is important for neuron vascular development. However, due to the highly divergent cytoplasmic domain, this integrin probably does not rely on inside-out signaling for affinity regulation. We therefore hypothesized that the β8 integrin uniquely assumes a constitutively high affinity state under physiological conditions. We discovered that β8 indeed exhibited high binding to soluble vitronectin in the presence of Ca2+ and the ligand binding could not be further enhanced by addition of Mn2+. The lower ectodomain stalk of the integrin, which is comprised by the integrin epidermal growth factor-like (I-EGF) domains and βTD domain, is critical for this high affinity conformation. In addition, we found that unlike other integrins, Mg2+ at low concentration inhibited β8 ligand binding. Mutagenesis studies indicated that β8 integrin possesses a unique cation binding site which might contribute to the ligand binding affinity. Our study showed that both the β8 lower ectodomain stalk and the head domain play an important role in its high affinity state under physiological conditions. J. Cell. Biochem. 118: 2044–2052, 2017. © 2016 Wiley Periodicals, Inc

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 α I and β 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 α and β transmembrane and cytoplasmic domains. © 2006 Elsevier Ltd. All rights reserved

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 α- and β-subunit dissociation in the integrin head-piece 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 α-subunit β-propeller domain and the β-subunit I-like domain. Basal and activation-dependent ligand binding by αIIbβ3 and αVβ3 was unaffected. The covalent linkage prevented dissociation of αIIbβ3 into its subunits on EDTA-treated cells. Whereas EDTA dissociated wild type αIIbβ3 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 αIIb and β3 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 αβ headpiece

Allosteric β\u3csub\u3e1\u3c/sub\u3e 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-β1 antibody, SG/19, that had been reported to inhibit the function of the β1 integrin on the cell surface. SG/19 recognized the wild type β1 subunit that exists in a conformational equilibrium between the high and low affinity states but bound poorly to a mutant β1 integrin that had been locked in a high affinity state. Epitope mapping of SG/19 revealed that Thr82 in the β1 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 α5β1 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 α5β1 integrin bound by SG/19 maintained a low affinity toward its physiological ligand fibronectin (Fn) whereas binding by function-blocking anti-α5 antibodies resulted in a complete loss of fibronectin binding. Thus a class of the anti-β 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

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 α and C-terminal end of β) of erythroid spectrin. The values of the inverse line width parameter (ΔH0-1) from a family of SpoI-1-368Δ peptides scanning residues 21-30 exhibited a periodicity of ∼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 ΔH0-1 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 α spectrin partial domain region. Mutations at these positions may lead to clinical symptoms. Similarly, the ΔH0-1 values from a family of spin-labeled SpβI-1898-2083Δ peptides also exhibited a periodicity of ∼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 ΔH0-1 values and a hydrophobic side with low ΔH0-1 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 αβ spectrin association at the tetramerization site. Mutations at some of these positions have been reported to be detrimental in clinical studies. © 2005 American Chemical Society