Crystal structure of the complete integrin alphaVbeta3 ectodomain plus an alpha/beta transmembrane fragment. J Cell Biol

Program in Leukocyte Biology and Inflammation, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 09/2009; 186(4):589-600. DOI: 10.1083/jcb.200905085
Source: PubMed


We determined the crystal structure of 1TM-alphaVbeta3, which represents the complete unconstrained ectodomain plus short C-terminal transmembrane stretches of the alphaV and beta3 subunits. 1TM-alphaVbeta3 is more compact and less active in solution when compared with DeltaTM-alphaVbeta3, which lacks the short C-terminal stretches. The structure reveals a bent conformation and defines the alpha-beta interface between IE2 (EGF-like 2) and the thigh domains. Modifying this interface by site-directed mutagenesis leads to robust integrin activation. Fluorescent lifetime imaging microscopy of inactive full-length alphaVbeta3 on live cells yields a donor-membrane acceptor distance, which is consistent with the bent conformation and does not change in the activated integrin. These data are the first direct demonstration of conformational coupling of the integrin leg and head domains, identify the IE2-thigh interface as a critical steric barrier in integrin activation, and suggest that inside-out activation in intact cells may involve conformational changes other than the postulated switch to a genu-linear state.

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Available from: Jose Luis Alonso
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    • "The integrin av chain was derived from the avb3 structure for modelling. The 3IJE structure of avb3 (the template structure used for modelling avb6) was resolved in a bent conformation under conditions that activate ligand binding in biochemical and cell biological assays (Xiong et al., 2009). The transmembrane-cytoplasmic domains of integrin aIIbb3 (PDB: 2KNC) (Yang et al., 2009) was the closest homologue, with a query coverage of 90% (identities: 10/14 (71%), positives: 10/14 (71%), gaps: 2/14 (14%)) and 77% (identities: 29/45 (64%), positives: 37/45 (82%), gaps: 0/45) for transmembrane and cytoplasmic domains of av and b6 respectively. "
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    • "Currently, the concept that the open integrin headpiece corresponds to the conformation with high affinity for ligand is well accepted for  1 ,  2 ,  6 , and  7 integrins, as referenced in the preceding paragraphs. However, this concept remains controversial in the  3 integrin field (Adair et al., 2005; Xiong et al., 2009) and has resulted in a lively dialog in the literature Figure 1. Integrin domain organization and conformational states. "
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    ABSTRACT: Carefully soaking crystals with Arg-Gly-Asp (RGD) peptides, we captured eight distinct RGD-bound conformations of the αIIbβ3 integrin headpiece. Starting from the closed βI domain conformation, we saw six intermediate βI conformations and finally the fully open βI with the hybrid domain swung out in the crystal lattice. The β1-α1 backbone that hydrogen bonds to the Asp side chain of RGD was the first element to move followed by adjacent to metal ion-dependent adhesion site Ca(2+), α1 helix, α1' helix, β6-α7 loop, α7 helix, and hybrid domain. We define in atomic detail how conformational change was transmitted over long distances in integrins, 40 Å from the ligand binding site to the opposite end of the βI domain and 80 Å to the far end of the hybrid domain. During these movements, RGD slid in its binding groove toward αIIb, and its Arg side chain became ordered. RGD concentration requirements in soaking suggested a >200-fold higher affinity after opening. The thermodynamic cycle shows how higher affinity pays the energetic cost of opening.
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    • "However, a single particle analysis of αVβ3 complexed with a recombinant fibronectin fragment has shown that αVβ3 can bind to a macromolecular ligand when it is in a bent state in the presence of Mn2+ [16]. The measurement of fluorescent energy transfer between the mAb bound to the β-propeller domain and the plasma membrane in live cells revealed that αVβ3 remains in a bent conformation when activated by Mn2+ or an activating mutation [17]. These lines of evidence suggest that the bent conformer is capable of binding not only small ligands, but also macromolecular ligands without undergoing substantial structural rearrangements of αVβ3 integrin. "
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