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dc.contributor.authorAlonso-García, Noelia-
dc.contributor.authorGarcía-Rubio, Inés-
dc.contributor.authorManso, José A.-
dc.contributor.authorBuey, Ruben M.-
dc.contributor.authorUrien, Hector-
dc.contributor.authorSonnenberg, Arnoud-
dc.contributor.authorJeschke, Gunnar-
dc.contributor.authorPereda, José M. de-
dc.identifier.citationEuromar 2014-
dc.descriptionResumen del trabajo presentado al Euromar celebrado en Zurich (Suiza) del 29 de junio al 3 de julio de 2014.-
dc.description.abstractThe integrin α6β4 is an essential component of hemidesmosomes, regulates keratinocyte proliferation and migration, and promotes carcinoma progression. The intracellular interactions of α6β4 are mediated mainly by the β4 subunit. In particular, the third and fourth fibronectin type-III domains (FnIII-3,4) of the β4 cytoplasmic moiety contain interaction sites for partner proteins BPAG1e and BP180, and participate in signaling events. Although the structure was unknown, the FnIII-3,4 was suggested to act as a hinge allowing the proximity of regions up- and downstream. In this contribution we report on the combination of different structural techniques to propose a model for the integrin β4 fragment FnIII-3,4. X-ray crystallography was used to resolve the structure of the individual FnIII-3 and FnIII-4 isolated domains at 1.6 and 1.5 Åresolution respectively. This technique provides atomic resolution but, since the complete fragment does not crystallize, is not able to supply any information about the relative orientation of the two domains or the structure of the twenty-three amino acid-long linker connecting them. Analysis of the FnIII-3,4 region by small angle x-ray scattering (SAXS) reveal that the whole fragment in solution adopts a compact, flat and slightly curved structure. However, low resolution prevents from identifying the interaction surfaces. Double Electron-Electron Resonance (DEER) experiments together with site-directed spin labeling of the fragment in solution allowed us to obtain thirteen distance constraints between residues located in the two different domains plus two distance constraints between a position in the linker and one of two FnIII domains. Combination of the distance constraints considering the FnIII domains to be rigid bodies yielded a bunch of atomic models at 2 Å. A number of random structures for the linker were generated into these structures taking into account bond length, bond angle, and Ramachandran constraints and only the ones compatible with the experimental constraints were preselected. Simulation of the SAXS experimental profile resulted in the final selection of a family of structures that revealed an evolutionary conserved surface extending along the two FnIII domains and the linker in the inner side of the crescent shape, which is likely to have an important structural or functional role. This case study shows that DEER, SAXS, X-Ray crystallography and molecular modeling can complement each other to finally obtain a plausible model for the structure of the whole protein fragment compatible with all the experimental results and constraints of the modeling.-
dc.titleCombination of DEER, SAXS, X-Ray crystallography and molecular modeling to obtain the structure of integrin ß4 domains-
dc.typeComunicación de congreso-
dc.description.versionPeer Reviewed-
Appears in Collections:(IBMCC) Comunicaciones congresos
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