@article {546756, title = {Complete integrin headpiece opening in eight steps.}, journal = {J Cell Biol}, volume = {201}, number = {7}, year = {2013}, pages = {1053-68}, abstract = { Carefully soaking crystals with Arg-Gly-Asp (RGD) peptides, we captured\ eight\ distinct RGD-bound conformations of the αIIbβ3integrin\ 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{\textquoteright} 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 {\r A} from the ligand binding site to the opposite end of the βI domain and 80 {\r A} 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. }, author = {Zhu, J. and Zhu, J. and Springer, T.A.} } @article {546771, title = {Domain 1 of mucosal addressin cell adhesion molecule has an I1-set fold and a flexible integrin-binding loop.}, journal = {J Biol Chem.}, volume = {288}, number = {9}, year = {2013}, pages = {6284-94}, abstract = {Mucosal\ addressin\ cell\ adhesion\ molecule\ (MAdCAM) binds integrin α4β7. Their interaction directs lymphocyte homing to mucosa-associated lymphoid tissues. The interaction between the two immunoglobulin superfamily (IgSF) domains of MAdCAM and integrin α4β7 is unusual in its ability to mediate either rolling\ adhesion\ or firm\ adhesion\ of lymphocytes on vascular surfaces. We determined four crystal structures of the IgSF domains of MAdCAM to test for unusual structural features that might correlate with this functional diversity. Higher resolution\ 1.7- and\ 1.4-A structures of the IgSF domains of MAdCAM in a previously described crystal lattice revealed two alternative conformations of the\ integrin-binding\ loop, which were deformed by large lattice contacts. New crystal forms in the presence of two different Fabs to MAdCAM demonstrate a shift in IgSF\ domain\ topology from the I2- to\ I1-set, with a switch ofintegrin-binding\ loop\ from CC{\textquoteright} to CD. The\ I1-set\ fold\ and CD\ loop\ appear biologically relevant. The different conformations seen in crystal structures suggest that the\ integrin-binding\ loop\ of MAdCAM is inherently\ flexible. This contrasts with rigidity of the corresponding loops in vascular\ cell\ adhesion\ molecule, intercellular\ adhesion\ molecule\ (ICAM)-1, ICAM-2, ICAM-3, and ICAM-5 and may reflect a specialization of MAdCAM to mediate both rolling and firm\ adhesion\ by binding to different α4β7 integrin conformations.}, author = {Yu, Y. and Zhu, J. and Huang, PS and Wang, J H and Pullen, N and Springer, T.A.} } @article {546781, title = {How Natalizumab Binds and Antagonizes α4 Integrins.}, journal = {J Biol Chem.}, volume = {288}, number = {45}, year = {2013}, pages = {32314-25}, abstract = {Natalizumab\ antibody to α4-integrins\ is used in therapy of multiple sclerosis and Crohn{\textquoteright}s disease. A crystal structure of the Fab bound to an α4 integrin β-propeller and thigh domain fragment shows that\ natalizumab\ recognizes human-mouse differences on the circumference of the β-propeller domain. The epitope is adjacent to but outside of a ligand-binding groove formed at the interface with the β-subunit βI domain and shows no difference in structure when bound to Fab. Competition between Fab and the ligand vascular cell adhesion molecule (VCAM) for binding to cell surface α4β1 shows noncompetitive antagonism. In agreement, VCAM docking models suggest that binding of domain 1 of VCAM to α4-integrins\ is unimpeded by the Fab, and that bound Fab requires a change in orientation between domains 1 and 2 of VCAM for binding to α4β1. Mapping of species-specific differences onto α4β1 and α4β7 shows that their ligand-binding sites are highly conserved. Skewing away from these conserved regions of the epitopes recognized by current therapeutic function-blocking antibodies has resulted in previously unanticipated mechanisms of action.}, author = {Yu, Y. and Sch{\"u}rpf, T. and Springer, T.A.} } @article {546786, title = {An internal ligand-bound, metastable state of a leukocyte integrin, αXβ2.}, journal = {J Cell Biol}, volume = {203}, number = {4}, year = {2013}, pages = {629-42}, abstract = {How is massive conformational change in integrins achieved on a rapid timescale? We report crystal structures of a\ metastable, putative transition\ state\ of\ integrin\ αXβ2. The αXβ2 ectodomain is bent; however, a lattice contact stabilizes its ligand-binding αI domain in a high affinity, open conformation. Much of the αI α7 helix unwinds, loses contact with the αI domain, and reshapes to form an\ internal\ ligand that binds to the interface between the β propeller and βI domains. Lift-off of the αI domain above this platform enables a range of extensional and rotational motions without precedent in allosteric machines. Movements of secondary structure elements in the β2 βI domain occur in an order different than in β3 integrins, showing that\ integrin\ β subunits can be specialized to assume different intermediate states between closed and open. Mutations demonstrate that the structure trapped here is\ metastable\ and can enable rapid equilibration between bent and extended-open\ integrin\ conformations and up-regulation of\ leukocyte\ adhesiveness.}, author = {Sen, M and Yuki, K and Springer, T.A.} } @article {546761, title = {Mechanisms by which von Willebrand disease mutations destabilize the A2 domain.}, journal = {J Biol Chem.}, volume = {288}, number = {9}, year = {2013}, pages = {6317-24}, abstract = {von\ Willebrand\ Factor (VWF) is an ultralong, concatameric, and adhesive glycoprotein. On short time scales, adhesiveness for platelets is activated by elongation of VWF by altered hydrodynamics at sites of hemostasis. Over longer time scales, the length of VWF is regulated by ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13), cleavage by which in the VWF\ A2\ domain\ is dependent on elongational force. Patients with\ von\ Willebrand\ disease\ type 2A present with increased bleeding due to\ mutations\ within the VWF\ A2\ domain\ that enhance cleavage. We tested using temperature and force the hypothesis that\ von\ Willebrand\ disease\ mutations\ disrupt\ A2\ force sensing by destabilizing the folded state.\ Mutations\ R1597W, M1528V, and E1638K reduced\ A2\ thermal stability by 10-18 {\textdegree}C. M1528V and E1638K showed a marked further decrease in stability upon calcium removal. In contrast, R1597W, which resides within the\ A2\ calcium-binding loop, exhibited similar stability in the presence and absence of calcium. Using single molecule optical tweezers and R1597W, we measured the force dependence of unfolding and refolding kinetics. In the presence of calcium, the R1597W mutation slowed the rate of refolding but had no effect on unfolding. The three\ mutations\ highlight the calcium-binding loop (R1597W), the hydrophobic core around the vicinal disulfide (M1528V), and hydrogen bonds to the α4-less loop (E1638K), as structural features critically important to the function of\ A2\ as a force sensor in regulating thrombogenic activity in the vasculature.}, author = {Xu, AJ and Springer, T.A.} } @article {546791, title = {Release of cellular tension signals self-restorative ventral lamellipodia to heal barrier micro-wounds.}, journal = {J Cell Biol}, volume = {201}, number = {3}, year = {2013}, pages = {449-65}, abstract = {Basic mechanisms by which\ cellular\ barriers sense and respond to integrity disruptions remain poorly understood. Despite its tenuous structure and constitutive exposure to disruptive strains, the vascular endothelium exhibits robust\ barrier\ function. We show that in response to micrometer-scale disruptions induced by transmigrating leukocytes, endothelial cells generate unique\ ventrallamellipodia\ that propagate via integrins toward and across these "micro-wounds" to close them. This novel actin remodeling activity progressively healed multiple\ micro-wounds\ in succession and changed direction during this process. Mechanical probe-induced micro-wounding of both endothelia and epithelia suggests that\ ventral\ lamellipodia\ formed as a response to force imbalance and specifically loss of isometric\ tension.\ Ventral\ lamellipodia\ were enriched in the Rac1 effectors cortactin, IQGAP, and p47Phox and exhibited localized production of hydrogen peroxide. Together with Apr2/3, these were functionally required for effective micro-wound healing. We propose that\ barrier\ disruptions are detected as local\ release\ of isometric\ tension/force unloading, which is directly coupled to reactive oxygen species-dependent\ self-restorative\ actin remodeling dynamics.}, author = {Martinelli R and Sage PT and Massol R and Varghese L and Sciuto T and Toporsian M and Dvorak AM and Kirchhausen T and Springer TA and Carman CV} }