Novel mutant human fibronectin FIII9-10 domain pair with increased conformational stability and biological activity.
The ninth and tenth type III domains (FIII9-10) in the central cell binding domain of human fibronectin contain integrin receptor binding sites, including RGD in FIII10 and a synergy site, PHSRN, in FIII9. The specific amino acids that contribute to cell binding have been identified by the use of wild-type and mutant fragments of human fibronectin containing the FIII9-10 domain pair. At high concentrations FIII9-10 mimics, to a large extent, the biological activity of the full-length fibronectin molecule. However, FIII9 is conformationally unstable, even in the context of the FIII9-10 pair. Here we report the construction of a series of hybrid mouse-human FIII9-10 pairs that confer varying degrees of conformational stability to FIII9. The conformational stability of the human FIII9 module was increased 2-3-fold by substitution of Leu1408 with Pro. We demonstrate that the biological activity of this mutant is enhanced. The resulting FIII9-10 mutant has good solution properties and will provide a template into which further mutations can be incorporated in order to probe the structure-function relationship of the cell binding module of fibronectin.
van der Walle
Christopher F
CF
Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Women's Centre, Level 3, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK.
Altroff
Harri
H
Mardon
Helen J
HJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D004926:Escherichia coli / Q000235:genetics; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007930:Leucine; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011392:Proline; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D012694:Serine; D012995:Solubility
D005353:Fibronectins; D011994:Recombinant Proteins; D012694:Serine; D011392:Proline; D007930:Leucine