Protein engineering of the hydrophobic domain of human factor IX

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Abstract
Vitamin K-dependent plasma proteins contain a highly conserved hydrophobic domain located between the γ-carboxyglutamic acid (Gla) domain and the first epidermal growth factor (EGF)-like domain. Here we have used protein engineering of the hydrophobic domain in human factor IX to investigate its function in intact factor IX. Mutant proteins were generated by site-directed mutagenesis and in vitro expression in Madin-Darby canine kidney (MDCK) cells. All of our mutants, including one with a deletion of the entire hydrophobic domain, were activated by factor XIa, showing that this domain is not required for factor IX activation. The results with the mutant Phe41 ← Val suggest that the hydrophobic domain interacts with the adjacent EGF-like domain. Our data for the Phe41 ← Asp mutant is consistent with, but cannot prove, a role for this residue in the maintenance of a phospholipid-binding structure required for factor IX function.

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