ABSTRACT
Talin is a large dimeric protein that couples integrins to cytoskeletal actin. Here, we report the structure of the C-terminal actin-binding domain of talin, the core of which is a five-helix bundle linked to a C-terminal helix responsible for dimerisation. The NMR structure of the bundle reveals a conserved surface-exposed hydrophobic patch surrounded by positively charged groups. We have mapped the actin-binding site to this surface and shown that helix 1 on the opposite side of the bundle negatively regulates actin binding. The crystal structure of the dimerisation helix reveals an antiparallel coiled-coil with conserved residues clustered on the solvent-exposed face. Mutagenesis shows that dimerisation is essential for filamentous actin (F-actin) binding and indicates that the dimerisation helix itself contributes to binding. We have used these structures together with small angle X-ray scattering to derive a model of the entire domain. Electron microscopy provides direct evidence for binding of the dimer to F-actin and indicates that it binds to three monomers along the long-pitch helix of the actin filament.
Subject(s)
Actins/metabolism , Recombinant Proteins/chemistry , Talin/chemistry , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , Dimerization , Image Processing, Computer-Assisted , Magnetic Resonance Spectroscopy , Mice , Microscopy, Electron, Scanning , Models, Biological , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Rabbits , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Talin/genetics , Talin/metabolismABSTRACT
The presence of anti-adenoviral serotype 5 (Ad5) antibodies may limit the efficacy of Ad5-mediated gene transfer. We therefore tested the hypothesis that intracoronary delivery of an adenovirus encoding human fibroblast growth factor type 4 (Ad5.FGF4) would improve regional myocardial function in an animal model of ischemia when high antibody levels preexist or after a prior intracoronary dose of Ad5. High anti-Ad5 antibody levels were generated in pigs by subcutaneous immunization with an adenovirus encoding LacZ (Ad5.lacZ). Neutralizing antibody levels increased 648-fold (range, 82- to 2108-fold) above preimmunization levels, and persisted for the duration of the study. Myocardial function during pacing-induced ischemia was improved in the ischemic region (p<0.001) at both 2 and 4 weeks after Ad5.FGF4 administration despite the presence of preexisting antibodies to Ad5. In a second set of experiments, the efficacy of a second intracoronary administration of Ad5 was determined by exposing the animals first to an intracoronary dose of Ad5.lacZ, followed 7 weeks later by the therapeutic dose of Ad5.FGF4. After delivery of Ad5.lacZ, anti-Ad5 antibody levels increased 8-fold (range, 1- to 18-fold), but this prior exposure to Ad5 did not prevent the therapeutic effects after subsequent intracoronary dosing with Ad5.FGF4 (p<0.001). In the porcine Ameroid constrictor model of myocardial ischemia the presence of anti-Ad5 antibodies or prior intracoronary dosing with adenovirus does not prevent the ability of Ad5.FGF4, delivered by intracoronary injection, from normalizing regional myocardial function.
