Structure-function analysis of Factor Seven Activating Protease (FSAP) by in-silico methods and its expression in insect cells.

Factor VII Activating Protease (FSAP) is a circulating serine protease in blood and is likely to be involved in regulating hemostasis and inflammatory processes in stroke. The zymogen form of FSAP (Pro-FSAP) is activated by charged molecules, including heparin and histones. Here, we tested the feasibility of FSAP expression in insect cells and analyzed its structure and evolution with in-silico methods. The expression of full-length (FL) FSAP and the serine protease domain (SPD) was very low, and this was slightly improved with the protease-inactivating mutant. The remainder of the protein, comprising the heavy chain (HC), was expressed at high levels and demonstrated binding to pro-FSAP activators, histones, and heparin. Since FL-FSAP was not satisfactorily expressible for structural studies, the AlphaFold structure was used for molecular dynamics simulations and in-silico docking studies with pro-FSAP activators. AlphaFold predicts a globular structure for the EGF, kringle and the trypsin domain as well as a disordered N-terminal region (NTR). Docking studies suggested that the EGF-3 domain interacts with heparin and the NTR with histones. Very likely, this disturbs the globular fold of the enzyme to expose the activation site and promotes autocatalytic activation. FSAP evolved in its current domain configuration in jawed vertebrates about 500 million years ago suggesting an important and conserved function. The insights from these studies will facilitate the expression of recombinant FSAP as well as enable a deeper understanding of its structure and biological functions in the context of stroke.