The third Trp-Lys-Ser (WKS) tripeptide motif in tissue factor is associated with a function site.
The tripeptide sequence Trp-Lys-Ser (WKS) is repeated three times in the extracellular ligand binding domain of human Tissue Factor (TF). Using site-directed mutagenesis, we replaced each of the WKS motifs in human TF by Arg-Lys-Gly (RKG), the least conserved replacement for the motif found in murine TF. This substitution in the first repeat W14KS, as well as a Trp14----Arg substitution, resulted in a structurally altered protein, whereas a conservative hydrophobic Trp14----Phe substitution resulted in a functionally normal protein. This suggests that Trp14 may contribute to a hydrophobic core rather than involvement of this motif in function. Replacement of the W45KS and W158KS motifs was associated with no detectable structural alterations; however, function was diminished with the RKG replacement of the third repeat. Mutant proteins with Lys159----Ala and Tyr157----Ala substitutions exhibited loss of function, whereas Tyr156----Ala and Ser160----Ala substitutions flanking the YWK sequence resulted in functional proteins. These data demonstrate that the W158KS motif in human TF is associated with a functional site and identify Lys159 in this motif as a functionally important residue.
Rehemtulla
A
A
Vascular Cell and Molecular Biology Program, Scripps Research Institute, La Jolla, CA 92037.
Ruf
W
W
Miles
D J
DJ
Edgington
T S
TS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005091:Exons / Q000235:genetics; D005110:Extracellular Space / Q000502:physiology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011817:Rabbits; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics* / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid; D013925:Thromboplastin / Q000737:chemistry / Q000235:genetics* / Q000502:physiology
D013925:Thromboplastin