Mutational mapping of functional residues in tissue factor: identification of factor VII recognition determinants in both structural modules of the predicted cytokine receptor homology domain.
Alanine scanning mutagenesis of tissue factor, the initiating receptor and cofactor molecule for the coagulation pathways, was used to define residue side chains with functional contributions. Approximately half of the residues were exchanged, and several stretches of functional residues throughout the entire extracellular domain were identified which contributed to overall coagulant function. Mutants were further characterized with respect to their affinity for binding of ligand, providing evidence that identified functional sequence spans are involved in ligand interaction. The tissue factor extracellular domain is suggested to adopt the folding pattern of the cytokine receptor homology unit, which is typically composed of two seven-beta-strand modules. Evaluation of the mutational analysis within this structural context suggests that functionally important residues are spatially proximate and clustered at the boundary of the predicted beta-strand modules. Residues contributing to ligand binding by tissue factor were identified in positions corresponding to ligand interactive residues in the growth hormone receptor and contact residues of other cytokine receptors, consistent with a conserved structural region for ligand interaction throughout the cytokine receptor family.
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Department of Immunology, Scripps Research Institute, La Jolla, California 92037.
Schullek
J R
JR
Stone
M J
MJ
Edgington
T S
TS
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D005167:Factor VII / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D017510:Protein Folding; D018121:Receptors, Cytokine / Q000737:chemistry*; D017385:Sequence Homology; D013329:Structure-Activity Relationship; D013925:Thromboplastin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D018121:Receptors, Cytokine; D005167:Factor VII; D013925:Thromboplastin; D000409:Alanine