Three-dimensional structural analysis of fibronectin heparin-binding domain mutations.
Using recombinant fibronectin proteins containing the V region and two point mutations in the high-affinity heparin-binding domain, we previously showed that these domains modulate tumor cell invasion as well as proteinase expression and apoptosis in human fibroblasts. Structurally, the wildtype counterparts to these two point mutations, together with four other discontinuous, positively charged residues, form a cationic cradle in domain III-13 of fibronectin that binds heparin. We constructed a three-dimensional model of this cationic cradle and determined whether the two engineered point mutations in the heparin-binding domain would alter this cradle conformation, thus explaining the altered cell behavior. Our model of fibronectin domain III-13 was generated from a template of the three-dimensional structure of a homologous (25% identity) domain, III-3, from tenascin. The amino acid sequences of III-13 that differed from tenascin III-3 were replaced, and side chains for positively charged arginines 6 and 7 were substituted with uncharged threonines. The model revealed that the two mutated threonine residues were solvent accessible, readily accommodated as part of an antiparallel beta strand, and remained part of the three-dimensional cradle. These models suggest that the two point mutations in the heparin-binding domain of fibronectin III-13 alter cell function probably through changes in charge and not through changes in the conformational structure of the cationic cradle. J. Cell. Biochem. Suppl. 36: 156-161, 2001.
Kapila
Y
Y
Department of Stomatology, University of California-San Francisco, San Francisco, CA 94143-0512, USA. ykapila@itsa.ucsf.edu
Doan
D
D
Tafolla
E
E
Fletterick
R
R
D000595:Amino Acid Sequence; D001665:Binding Sites; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D005353:Fibronectins; D011994:Recombinant Proteins; D006493:Heparin