All six modules of the gelatin-binding domain of fibronectin are required for full affinity.
The gelatin-binding sites of fibronectin are confined to a 42-kDa region having four type I and two type II modules in the following order: I(6)-II(1)-II(2)-I(7)-I(8)-I(9). To determine the relative importance of each module for recognition of gelatin, recombinant green fluorescent fusion proteins were prepared in which individual modules or groups of modules were deleted, and the resulting proteins were tested for binding to gelatin by analytical affinity chromatography. Deletion of both type II modules did not eliminate binding, confirming that at least some of the type I modules in this region are able to bind gelatin. It was found that deletion of type I module 6 tends to increase the affinity, whereas deletion of any other module decreases it. Deletion of module I(9) had a large effect but only if module II(2) was also present, suggesting an interaction between these two noncontiguous modules. Analysis of more than 20 recombinant fusion products led to the conclusion that all modules contribute to the interaction either directly by contacting the ligand or indirectly through module-module interactions.
Katagiri
Yasuhiro
Y
Biochemistry Department, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland 20855, USA.
Brew
Shelesa A
SA
Ingham
Kenneth C
KC
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005780:Gelatin / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012685:Sepharose / Q000494:pharmacology; D018411:Spodoptera
D005353:Fibronectins; D007202:Indicators and Reagents; D008024:Ligands; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005780:Gelatin; D012685:Sepharose