title: Studies of the local conformational properties of the cell-adhesion domain of collagen type IV in synthetic heterotrimeric peptides.
abstract: Collagen type IV is a specialized form of collagen that is found only in basement membranes. It is involved in integrin-mediated cell-adhesion processes, and the responsible binding sites for the alpha1beta1 integrin cell receptor have been identified as Asp461 of the two alpha1 chains and Arg461 of the alpha2 chain. In the most plausible stagger of native collagen type IV the alpha2 chain is the tailing one. This has recently been confirmed by the differentiated binding affinities of synthetic heterotrimeric collagen peptides in which the chains were staggered in this native register as well as in the less plausible alpha1alpha2alpha1' register with an artificial cystine knot. In the present work, two heterotrimeric collagen peptides with chain registers identical to the previous ones were synthesized for fluorescence resonance energy transfer and emission anisotropy measurements, exploiting the native Phe464 in the alpha2 chain as donor and an Ile467Tyr mutation in the alpha1' chain as acceptor fluorophore. This fluorophore pair allowed extraction of more detailed information on the conformational properties of the cell-adhesion epitope incorporated into the central part of the trimeric collagen model peptides. A comparison of the experimentally derived values of the interfluorophore distance and of the orientation factor kappa(2) with the values extracted from the molecular model of the trimer in the native stagger confirmed a triple-helical structure of the adhesion-site portion at low temperature. The thermal unfolding of this central domain was specifically monitored by emission anisotropy, allowing unambiguous assignment of the three structural domains of the trimeric collagen molecules detected by microcalorimetry, with the integrin binding site as the portion of weakest triple-helical stability flanked by two more stable triple-helical regions. The results are consistent with the picture of a conformational microheterogeneity as the responsible property for selective recognition of collagens by interacting proteins.
authors.affiliation: Max-Planck-Institute for Biochemistry, Am Klopferspitz 18A, D-82152 Martinsried, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Saccà
authors.forename: Stella
authors.initials: S
authors.lastname: Fiori
authors.forename: Luis
authors.initials: L
authors.lastname: Moroder
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018821:CD18 Antigens / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D002942:Circular Dichroism; D024141:Collagen Type IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D066298:In Vitro Techniques; D039423:Integrin alpha1 / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D018821:CD18 Antigens; D024141:Collagen Type IV; D039423:Integrin alpha1; D011994:Recombinant Proteins