Towards atomistic characterization of blood coagulation proteins by NMR spectroscopy

Proteins involved in the blood coagulation cascade are of vital biomedical importance, and most essential blood-clotting reactions occur preferentially on phospholipid membranes. Understanding the interactions between the proteins and membranes involved in blood clotting is crucial to the continued development of successful treatments for blood coagulation disorders such as hemophilia, von Willebrand disease, and thrombosis. In normal hemostasis, the blood-clotting cascade is initiated when factor VIIa (fVIIa, other clotting factors are named similarly) binds to human tissue factor (TF), a 29 kDa integral membrane protein. The TF/fVIIa complex in turn activates fX and fIX, eventually concluding with clot formation. Several X-ray crystal structures of the extracellular domain of TF (sTF) exist; however, some of these structures are lacking electron density in functionally important dynamic loops that can be accessed using nuclear magnetic resonance (NMR) spectroscopy. Here, the nanocrystalline and membrane-bound forms of TF are investigated with solid-state NMR and compared to the previously published solution NMR sTF assignments. We have prepared several samples of the sTF with uniform (U)-13C, 15N labeling and a variety of 13C sparse labeling schemes, precipitated with polyethylene glycol and ammonium sulfate. A suite of interresidue and intraresidue multidimensional solid-state NMR experiments have been acquired in order to complete the chemical shift assignments of microcrystalline sTF and compare the effects of different precipitation agents on chemical shifts, focusing on the loop residues missing in the X-ray crystal structures. The dynamics of TF has also been investigated using solution NMR relaxation experiments, solid-state T-MREV experiments to extract order parameters, and molecular dynamics simulations. (U)-13C, 15N mTF samples, a construct that includes the transmembrane helix, have been incorporated into both phospholipid Nanodiscs and POPC/DPPC liposomes. The impact of different lipid preparations on the structure of TF as well as mTF functional assay data are presented. Our preliminary results indicate that TF retains its primarily beta-sheet secondary structure upon membrane binding; however, perturbations are observed in several flexible loops near the membrane surface that have been shown to be necessary for enzymatic activity. These studies provide a deeper understanding of the structure and mechanism of the vital blood coagulation protein TF in atomistic detail. Here, the nanocrystalline and membrane-bound forms of TF are investigated with solid-state NMR (SSNMR) and compared to the previously published solution NMR sTF assignments (1). We have prepared several samples of the sTF with uniform (U)-13C, 15N labeling and a variety of 13C sparse labeling schemes (2), precipitated with polyethylene glycol and ammonium sulfate. A suite of interresidue and intraresidue multidimensional SSNMR experiments have been acquired in order to complete the chemical shift assignments of microcrystalline sTF and compare the effects of different precipitation agents on chemical shifts, focusing on the loop residues missing in the X-ray crystal structures. The dynamics of TF has also been investigated using solution NMR relaxation experiments, solid-state T-MREV experiments to extract order parameters, and molecular dynamics simulations. (U)-13C, 15N mTF samples, a construct that includes the transmembrane helix, have been incorporated into both phospholipid Nanodiscs and POPC/DPPC liposomes. The impact of different lipid preparations on the structure of TF as well as mTF functional assay data will be presented. Our preliminary results indicate that TF retains its primarily beta-sheet secondary structure upon membrane binding; however, perturbations are observed in several flexible loops near the membrane surface that have been shown to be necessary for enzymatic activity. These studies will provide a deeper understanding of the structure and mechanism of the vital blood coagulation protein TF at atomistic detail