Mapping early conformational changes in αIIb and β3 during biogenesis reveals a potential mechanism for αIIbβ3 adopting its bent conformation

Abstract

Current evidence supports a model in which the low-affinity state of the platelet integrin ␣IIb␤3 results from ␣IIb␤3 adopting a bent conformation. To assess ␣IIb␤3 biogenesis and how ␣IIb␤3 initially adopts the bent conformation, we mapped the conformational states occupied by ␣IIb and ␤3 during biogenesis using conformation-specific monoclonal antibodies (mAbs). We found that ␣IIb␤3 complex formation was not limited by the availability of either free pro-␣IIb or free ␤3, suggesting that other molecules, perhaps chaperones, control complex formation. Five ␤3-specific, ligand-induced binding site (LIBS) mAbs reacted with much or all free ␤3 but not with ␤3 when in complex with mature ␣IIb, suggesting that ␤3 adopts its mature conformation only after complex formation. Conversely, 2 ␣IIb-specific LIBS mAbs directed against the ␣IIb Calf-2 region adjacent to the membrane reacted with only minor fractions of free pro-␣IIb, raising the possibility that pro-␣IIb adopts a bent conformation early in biogenesis. Our data suggest a working model in which pro-␣IIb adopts a bent conformation soon after synthesis, and then ␤3 assumes its bent conformation by virtue of its interaction with the bent pro-␣IIb. IntroductionIntegrin receptors are composed of heterodimers of ␣ and ␤ subunits. The ␤3 family of integrin receptors is composed of ␣IIb␤3, which is specific for platelets and their precursor megakaryocytes, and ␣v␤3, which is more widely distributed on many cells, including osteoclasts and endothelial cells. 1 ␣IIb␤3 plays an important role in platelet aggregation, and inhibitors of the receptor have demonstrated efficacy in preventing thrombotic complications of percutaneous coronary interventions. 2 Previous studies of ␣IIb␤3 integrin biogenesis have provided important information on the synthesis of the ␣IIb and ␤3 subunits, ␣IIb␤3 complex formation, carbohydrate processing, transport of the assembled complexes from the endoplasmic reticulum (ER) to the Golgi, cleavage of ␣IIb into heavy and light chains in the Golgi, and subsequent transport of the mature receptor to granule and platelet surface membranes. [3][4][5][6][7][8][9][10][11] In particular, they suggested that the availability of either free pro-␣IIb or free ␤3 limits ␣IIb␤3 complex formation. 7,9 The crystal structure of the extracellular domain of ␣v␤3 unexpectedly revealed that the receptor had a bent conformation, 12 and electron microscopy suggested that activation and ligand binding are associated with the adoption of an extended conformation. 13 It is presumed that ␣IIb␤3 also undergoes a transformation from a bent to an extended conformation on activation, 14 which occurs when platelets are stimulated with one or more agonists, resulting in binding of ligands, including fibrinogen. The crystal structure of the headpiece of ␣IIb␤3 in complex with ligandmimetics also identified a swing-out motion of the ␤3 hybrid domain at its juncture with the ␤A (I-like) domain that is thought to contribute to ligand binding and/or the initiation of outside-in sig...

The GPIIb/IIIa (integrin αIIbβ3) odyssey: a technology-driven saga of a receptor with twists, turns, and even a bend

“…Adapted from Qin et al 143 The molecular models of ␣IIb␤3 were constructed using MODELLER 8v2 and the PDBs ITY6, IU8C, and IYUK as previously described. 241 I-EGF, integrin epidermal growth factor domain; ␤-TD, ␤-terminal domain. For personal use only.…”

Intact αIIbβ3 Integrin Is Extended after Activation as Measured by Solution X-ray Scattering and Electron Microscopy

“…Previous x-ray crystallography studies on the ␣ IIb ␤ 3 , ␣ V ␤ 3 , and ␣ x ␤ 2 ectodomains showed that resting integrins are in a compact, bent conformation (8 -10). In addition, global structural rearrangements upon activation were demonstrated by electron microscopy (EM) of soluble ectodomain truncations and supported by exposure of activation epitopes known as ligand-induced binding sites (LIBSs), 3 which are not accessible in the resting state (11)(12)(13)(14)(15). Two distinct conformational changes are seen, extension and headpiece opening (hybrid domain swing-out), resulting in three overall conformations: bent with a closed headpiece, extended with a closed headpiece, and extended with an open headpiece that is stabilized by ligand binding (11,16).…”

Effects of Limiting Extension at the αIIb Genu on Ligand Binding to Integrin αIIbβ3

“…An energy optimized model of the complete extracellular domain of ␣IIb␤3 was built to identify candidate regions of the molecule that might selectively restrict ␣IIb extension around the genu. As previously described (32), the conformation of the ␣IIb chain of the bent, inactive, unliganded ␣IIb␤3 was generated with MODELLER 8v2 (33) using the coordinates of the ␤ propeller from the crystal structure of ␣IIb␤3 (residues 1-453; Protein Data Bank entry 1TY6 (11)) and the coordinates of ␣V for the remainder of the sequence (Protein Data Bank entry 1U8C (34)) Most of the coordinates of the ␤3 chain of the bent, inactive, unliganded ␣IIb␤3 conformation were extracted from the ␣V␤3 complex (Protein Data Bank entry 1U8C), whereas the missing domains, IEGF-1 (435-475) and IEGF-2 (486 -522), were constructed with MODELLER 8v2 using the crystal structures of the ␤2 IEGF-1 (Protein Data Bank entry 1YUK (35)) and the ␣V␤3 IEGF-3 (Protein Data Bank entry 1U8C) domains, respectively, as templates. A model of the extended conformation of ␣IIb␤3 based on changes around the ␣IIb genu and ␤3 IEGF-1 and 2 domains was generated from our energy-minimized model of the complete extracellular unliganded bent conformation of ␣IIb␤3 by a 26°rotation of the ␣IIb 601-960 and ␤3 480 -690 regions around the C␣-N bond of ␣IIb residue 600, followed by a 22°rotation of the ␣IIb 599 -960 and ␤3 480 -690 regions around the C␣-C␣ axis of ␣IIb residues 607-608.…”