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.


Available from: Jose Luis Alonso
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Integrin αvβ6 is an epithelially-restricted heterodimeric transmembrane glycoprotein, known to interact with the urokinase plasminogen activating receptor (uPAR), playing a critical role in cancer progression. While the X-ray crystallographic structures of segments of other integrin heterodimers are known, there is no structural information for the complete αvβ6 integrin to assess its direct interaction with uPAR. We have performed structural analysis of αvβ6•uPAR interactions using model data with docking simulations to pinpoint their interface, in accord with earlier reports of the β-propeller region of integrin α-chain interacting with uPAR. Interaction of αvβ6•uPAR was demonstrated by our previous study using immunoprecipitation coupled with proteomic analysis by mass spectrometry. Recently this interaction was validated with proximity ligation assays and peptide arrays. The data suggested that two potential peptide regions from domain II and one peptide region from domain III of uPAR, interact with αvβ6 integrin. Only the peptide region from domain III is consistent with the three-dimensional interaction site proposed in this study. The molecular basis of integrin αvβ6•uPAR binding using structural data is discussed for its implications as a potential therapeutic target in cancer management.
    Journal of Structural Biology 01/2014; 185(3). DOI:10.1016/j.jsb.2014.01.001 · 3.23 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    The Journal of Cell Biology 06/2013; 201(7):1053-1068. DOI:10.1083/jcb.201212037 · 9.83 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Epitopes for a panel of anti-αVβ3 monoclonal antibodies (mAbs) were investigated to explore the activation mechanism of αVβ3 integrin. Experiments utilizing αV/αIIb domain-swapping chimeras revealed that among the nine mAbs tested, five recognized the ligand-binding β-propeller domain and four recognized the thigh domain, which is the upper leg of the αV chain. Interestingly, the four mAbs included function-blocking as well as non-functional mAbs, although they bound at a distance from the ligand-binding site. The epitopes for these four mAbs were further determined using human-to-mouse αV chimeras. Among the four, P3G8 recognized an amino acid residue, Ser-528, located on the side of the thigh domain, while AMF-7, M9, and P2W7 all recognized a common epitope, Ser-462, that was located close to the α-genu, where integrin makes a sharp bend in the crystal structure. Fibrinogen binding studies for cells expressing wild-type αVβ3 confirmed that AMF-7, M9, and P2W7 were inhibitory, while P3G8 was non-functional. However, these mAbs were all unable to block binding when αVβ3 was constrained in its extended conformation. These results suggest that AMF-7, M9, and P2W7 block ligand binding allosterically by stabilizing the angle of the bend in the bent conformation. Thus, a switchblade-like movement of the integrin leg is indispensable for the affinity regulation of αVβ3 integrin.
    PLoS ONE 06/2013; 8(6):e66096. DOI:10.1371/journal.pone.0066096 · 3.23 Impact Factor
Show more