Integrin αIIbβ3 in a Membrane Environment Remains the Same Height after Mn2+ Activation when Observed by Cryoelectron Tomography

Self Cite

Background:Integrins undergo large conformational changes when ligand-bound. Results: Using nanodisc technology and EM, we observed Mn 2ϩ -activated ␣ IIb ␤ 3 integrin both alone and fibrin-bound. Conclusion: MnCl 2 -activated ␣ IIb ␤ 3 integrin alone has a compact conformation, becoming fully upright and open when fibrin-bound. Significance: The first structure of membrane-embedded integrins bound to physiological substrate reveals the importance of integrin extension in macromolecular ligand binding.

Section: Resultssupporting

confidence: 92%

“…Preparation of Integrin Nanodiscs and Two-dimensional Fibrin Complexes-Full-length ␣ IIb ␤ 3 integrin was purified from outdated blood platelets following published procedures (30,32,33). Integrin nanodiscs were assembled using a protocol adapted previously (34,35).…”

Section: Methodsmentioning

confidence: 99%

The Structure of a Full-length Membrane-embedded Integrin Bound to a Physiological Ligand

Dai

Taylor

et al. 2015

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“…So it was observed by sedimentation equilibrium that fibrinogen in the presence of Ca 2ϩ and Mn 2ϩ did not bind to ␣IIb␤3 (K D Ͼ 1 mM) (40), and the attempts to isolate ␣IIb␤3-fibrinogen complexes in detergent solution at physiological concentrations of fibrinogen by size exclusion chromatography, in the presence of Mn 2ϩ or the Fab of an anti-␣IIb␤3 antibody, which induces platelet activation, fibrinogen binding, and platelet aggregation, always failed. 4 Equally, the height of ␣IIb␤3 reconstituted in liposomes does not change in the presence of Mn 2ϩ , as observed by cryoelectron tomography (14), and preliminary results of SANS experiments suggest that the binding of the Fab fragments of one of these monoclonal antibodies to ␣IIb␤3 in detergent solution is not followed by the extension of the integrin structure. 3 So to clear the conflicting experimental observations on the Mn 2ϩ activation of ␣IIb␤3 and on whether its activated state is in the bent or the extended configuration, the reconstitution of the integrin in single bilayer vesicles made using deuterated lipids and its activation using Mn 2ϩ or an Fab of an anti-␣IIb␤3 activating antibody, in the absence or in the presence of equimolecular fibrinogen concentration, is one of the first experiments to do to try to make progress in this direction.…”

Section: Discussionmentioning

confidence: 81%

“…In addition, the soluble Mn 2ϩ -bound r-␣V␤3 ectodomain, the complex of this ectodomain with a fibronectin fragment (with type III domains 7-10 and the extradomain B), and Mn 2ϩ -activated ␣IIb␤3 were equally in the bent configuration, as determined by electron microscopy (EM) 2 (13,14). Finally, ␣IIb␤3 in resting and physiologically activated platelets were also in the bent configuration, as found by epitope mapping and competition among monoclonal antibodies directed to the C-terminal region of the ␣IIb subunit and to the N-terminal domain of the ␤3 subunit (9) and by fluorescence intramolecular energy transfer (15).…”

mentioning

confidence: 99%

Three-dimensional Model of Human Platelet Integrin αIIbβ3 in Solution Obtained by Small Angle Neutron Scattering

Nogales

García

Pérez

et al. 2010

Integrin ␣IIb␤3 is the major membrane protein and adhesion receptor at the surface of blood platelets, which after activation plays a key role in platelet plug formation in hemostasis and thrombosis. Small angle neutron scattering (SANS) and shape reconstruction algorithms allowed formation of a low resolution three-dimensional model of whole ␣IIb␤3 in Ca 2؉ /detergent solutions. Model projections after 90°rotation along its long axis show an elongated and "arched" form (135°) not observed before and a "handgun" form. This 20-nm-long structure is well defined, despite ␣IIb␤3 multidomain nature and expected segmental flexibility, with the largest region at the top, followed by two narrower and smaller regions at the bottom. Docking of this SANS envelope into the high resolution structure of ␣IIb␤3, reconstructed from crystallographic and NMR data, shows that the solution structure is less constrained, allows tentative assignment of the disposition of the ␣IIb and ␤3 subunits and their domains within the model, and points out the structural analogies and differences of the SANS model with the crystallographic models of the recombinant ectodomains of ␣IIb␤3 and ␣V␤3 and with the cryo-electron microscopy model of whole ␣IIb␤3. The ectodomain is in the bent configuration at the top of the model, where ␣IIb and ␤3 occupy the concave and convex sides, respectively, at the arched projection, with their bent knees at its apex. It follows the narrower transmembrane region and the cytoplasmic domains at the bottom end. ␣IIb␤3 aggregated in Mn 2؉ /detergent solutions, which impeded to get its SANS model.Blood platelets have distinct mechanisms to respond to the different agonists of platelet activation, all of which end up with the acquisition by the integrin ␣IIb␤3 or glycoprotein IIb/IIIa of the capacity to recognize and bind fibrinogen and other adhesive proteins. Binding of fibrinogen, the major adhesive protein in plasma, to activated ␣IIb␤3 and the binding of fibronectin, the major adhesive protein in the extracellular matrix of subendothelium, to resting or activated ␣IIb␤3 lead to the formation of interplatelet cross-linking and to platelet adhesion to the subendothelium, respectively, and, eventually, to the formation of the platelet plug. Thus, knowledge of the fibrinogen receptor and the mechanisms of induction and modulation of its activity is determinant to understand hemostasis and the pathophysiological paths leading to thrombosis, as well as to develop new ways to prevent, detect, and treat thrombosis effectively, securely, and selectively. After 20 years of molecular genetics and 10 years of high resolution structural biology of integrins, the disposition of an integrin in the membrane is unknown, the modifications in the cytoplasmic domains and the transmembrane segments related with the receptor activation are still not definitively defined, and the molecular mechanism of activation and molecular properties of the activated receptor are still under discussion (1-4). Structural and cell biological informat...

“…3B, Mn 2ϩ seems to lessen the requirement for integrin extension for Fbg binding. Mn 2ϩ activation alone has consistently been reported not to be accompanied by integrin extension (30 . These results may explain why ligand binding was observed for the bent conformer in some studies in which Mn 2ϩ was utilized to induce ligand binding (13,14).…”

Section: Discussionmentioning

confidence: 98%

Structural Requirements for Activation in αIIbβ3 Integrin

Kamata

Handa

Ito

et al. 2010

Integrins are postulated to undergo structural rearrangement from a low affinity bent conformer to a high affinity extended conformer upon activation. However, some reports have shown that a bent conformer is capable of binding a ligand, whereas another report has shown that integrin extension does not absolutely lead to activation. To clarify whether integrin affinity is indeed regulated by the so-called switchblade-like movement, we have engineered a series of mutant ␣IIb␤3 integrins that are constrained specifically in either a bent or an extended conformation. These mutant ␣IIb␤3 integrins were expressed in mammalian cells, and fibrinogen binding to these cells was examined. The bent integrins were created through the introduction of artificial disulfide bridges in the ␤-head/␤-tail interface. Cells expressing bent integrins all failed to bind fibrinogen unless pretreated with DTT to disrupt the disulfide bridges. The extended integrins were created by introducing N-glycosylation sites in amino acid residues located close to the ␣-genu, where the integrin legs fold backward. Among these mutants, activation was maximized in one integrin with an N-glycosylation site located behind the ␣-genu. This extension-induced activation was completely blocked when the swing-out of the hybrid domain was prevented. These results suggest that the bent and extended conformers represent low affinity and high affinity conformers, respectively, and that extension-induced activation depends on the swing-out of the hybrid domain. Taken together, these results are consistent with the current hypothesis that integrin affinity is regulated by the switchblade-like movement of the integrin legs.Integrin-mediated bidirectional signaling is closely associated with the structural rearrangement of integrin itself. During inside-out signaling, talin has been shown to bind to the ␤ cytoplasmic tail and to disrupt the endogenous interaction between the ␣ and ␤ cytoplasmic tails (1, 2). The dissociation of the two tails induces a structural rearrangement of the extracellular domains increasing the affinity to the ligand. During outside-in signaling, ligand binding in turn induces the structural rearrangement of the extracellular domains. This structural change propagates through the plasma membrane to separate the cytoplasmic tails, providing binding sites for numerous cytoplasmic proteins (3). Thus, the two structures flanking the plasma membrane affect each other. This structural rearrangement can be detected using a group of monoclonal antibodies (mAbs) that bind preferentially to the ligand-bound form (4). These anti-LIBS 2 (ligand-induced binding site) mAbs have been used not only to investigate the activation status of a specific integrin but also to activate it (5). However, without information on the actual three-dimensional structure, it is impossible to determine the specific conformation recognized by each anti-LIBS mAb.The first observation of the actual three-dimensional structure of integrin was made using conventional electron ...