THE 3RD 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.