6r2w
Crystal structure of the super-active FVIIa variant VYT in complex with tissue factor
Structural highlights
FunctionTF_HUMAN Initiates blood coagulation by forming a complex with circulating factor VII or VIIa. The [TF:VIIa] complex activates factors IX or X by specific limited protolysis. TF plays a role in normal hemostasis by initiating the cell-surface assembly and propagation of the coagulation protease cascade.[1] Publication Abstract from PubMedTwo decades of research have uncovered the mechanism by which the complex of tissue factor (TF) with the plasma serine protease factor VIIa (FVIIa) mediates the initiation of blood coagulation. Membrane-anchored TF directly interacts with substrates and induces allosteric effects in the protease domain of FVIIa. These properties are also recapitulated by the soluble ectodomain of TF (sTF). At least two interdependent allosteric activation pathways originate at the FVIIa:sTF interface and are proposed to enhance FVIIa activity upon sTF binding. Here, we sought to engineer an sTF-independent FVIIa variant by stabilizing both proposed pathways, with one pathway terminating at segment 215-217 in the activation domain and the other pathway terminating at the N-terminus insertion site. To stabilize segment 215-217, we replaced the flexible 170 loop of FVIIa by the more rigid 170 loop from trypsin and combined it with an L163V substitution (FVIIa-VYT). The FVIIa-VYT variant exhibited 1.8-fold higher amidolytic activity than the FVIIa:sTF complex, and both displayed similar FX activation and antithrombin inhibition kinetics. The sTF-independent activity of FVIIa-VYT was partly mediated by an increase in the N-terminus insertion and, as shown by X-ray crystallography, partly by Tyr-172 inserting into a cavity in the activation domain stabilizing the S1 substrate-binding pocket. The combination with L163V likely drove additional changes in a delicate hydrogen-bonding network that further stabilized S1-S3 sites. In summary, we report the first FVIIa variant that is catalytically independent of sTF and provide evidence supporting the existence of two TF-mediated allosteric activation pathways. Beating tissue factor at its own game: Design and properties of a soluble tissue factor-independent coagulation factor VIIa.,Sorensen AB, Tuneew I, Svensson LA, Persson E, Ostergaard H, Overgaard MT, Olsen OH, Gandhi PS J Biol Chem. 2019 Dec 4. pii: RA119.009183. doi: 10.1074/jbc.RA119.009183. PMID:31801825[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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