High-Resolution NMR Studies of Human Tissue Factor

<div><p>In normal hemostasis, the blood clotting cascade is initiated when factor VIIa (fVIIa, other clotting factors are named similarly) binds to the integral membrane protein, human tissue factor (TF). The TF/fVIIa complex in turn activates fX and fIX, eventually concluding with clot formation. Several X-ray crystal structures of the soluble extracellular domain of TF (sTF) exist; however, these structures are missing electron density in functionally relevant regions of the protein. In this context, NMR can provide complementary structural information as well as dynamic insights into enzyme activity. The resolution and sensitivity for NMR studies are greatly enhanced by the ability to prepare multiple milligrams of protein with various isotopic labeling patterns. Here, we demonstrate high-yield production of several isotopically labeled forms of recombinant sTF, allowing for high-resolution NMR studies both in the solid and solution state. We also report solution NMR spectra at sub-mM concentrations of sTF, ensuring the presence of dispersed monomer, as well as the first solid-state NMR spectra of sTF. Our improved sample preparation and precipitation conditions have enabled the acquisition of multidimensional NMR data sets for TF chemical shift assignment and provide a benchmark for TF structure elucidation.</p></div

Components of media used for isotopically labeled growths of sTF.

<p>Components of media used for isotopically labeled growths of sTF.</p

Solid-state ¹⁵N-¹³CA correlation spectra of uniform and glycerol labeled sTF.

<p>(A) ¹⁵N-¹³CA correlation spectrum of ~30 mg of uniform ¹³C,¹⁵N ammonium sulfate-precipitated sTF with 5 mM Cu-EDTA. The spectrum was acquired for 3 hours and 20 minutes with an acquisition time of 25 ms. (B) ¹⁵N-¹³CA correlation spectrum of ~25 mg of 2-¹³C glycerol, ²H, ¹⁵N ammonium sulfate-precipitated sTF with 5 mM Cu-EDTA. The spectrum was acquired for 3 hours with an acquisition time of 30 ms. Both spectra were collected at 750 MHz (¹H frequency) at a variable temperature set point of -5°C with a MAS rate of 12.5 kHz. Both spectra were processed with 10 Hz of Lorentzian-to-Gaussian line broadening in each dimension, sine bell apodization, and zero filled 8192 points in the direct (F1) dimension and 2048 in the indirect (F2) dimension with contours drawn at 6 times the noise floor. (C) ¹³C 1D slices extracted from the ¹⁵N-¹³CA 2D spectrum of uniform ¹³C-¹⁵N ammonium sulfate-precipitated sTF shown in A. The corresponding ¹⁵N chemical shifts in the indirect dimension are indicated. (D) ¹³C 1D slices extracted from the ¹⁵N-¹³CA 2D spectrum of 2-¹³C glycerol, ²H, ¹⁵N ammonium sulfate-precipitated sTF shown in B. The corresponding ¹⁵N chemical shifts in the indirect dimension are indicated.</p

¹H-¹⁵N 2D HSQC correlation spectrum.

<p>Solution NMR fingerprint spectrum of 100 μM uniform ¹³C,¹⁵N sTF in 50 mM sodium phosphate (pH 6.5), 50 mM NaCl, 5% DSS, 10% D₂O, 0.1% NaN₃ acquired at a variable temperature set point of 35°C on a Varian/Agilent VNMRS 17.6 T (750 MHz ¹H frequency) spectrometer.</p

SDS-PAGE analysis of sTF purification.

<p>SDS-PAGE 12% acrylamide gel of sTF samples stained with Coomassie Brilliant Blue R-250 showing: Lane 1: Precision Plus Protein^TM Dual Color marker (Bio-Rad, Hercules, CA, USA); Lane 2: water and sucrose combined supernatant; Lane 3: Q-Sepharose® Fast Flow supernatant; Lane 4: Post 0.45 μm filter; Lane 5: pre-load on Ni²⁺ affinity column; Lane 6: 500 mM imidazole elution; Lane 7: concentrated sTF.</p

Solid-state ¹³C-¹³C 2D correlation spectra of uniform ¹³C,¹⁵N sTF.

<p>(A) ¹³C-¹³C 2D correlation spectrum with 25 ms of DARR mixing of ~15 mg uniform ¹³C,¹⁵N sTF precipitated with 15% PEG 3350 acquired at 750 MHz (¹H frequency) for 12 hours with a MAS rate of 12.5 kHz at a variable temperature set point of 0°C. (B) ¹³C-¹³C 2D correlation spectrum with 50 ms of DARR mixing of ~31 mg uniform ¹³C,¹⁵N sTF precipitated with 1.8 M ammonium sulfate and 5 mM Cu-EDTA acquired at 750 MHz (¹H frequency) for 24 hours with a MAS rate of 12.5 kHz at a variable temperature set point of -5°C. Both spectra were processed with 20 Hz of Lorentzian-to-Gaussian line broadening in each dimension, sine bell apodization, and zero filled 8192 points in the direct (F1) dimension and 4096 in the indirect (F2) dimension with contours drawn at 6 times the noise floor. (C) ¹³C 1D slices extracted from the ¹³C-¹³C 2D spectrum of PEG-precipitated sTF shown in A. The corresponding ¹³C chemical shifts in the indirect dimension are indicated. (D) ¹³C 1D slices extracted from the ¹³C-¹³C 2D spectrum of ammonium sulfate-precipitated sTF shown in B. The corresponding ¹³C chemical shifts in the indirect dimension are indicated.</p