Structural Architecture of Prothrombin in Solution Revealed by Single Molecule Spectroscopy

Abstract

The coagulation factor prothrombin has a complex spatial organization of its modular assembly that comprises the N-terminal Gla domain, kringle-1, kringle-2, and the C-terminal protease domain connected by three intervening linkers. Here we use single molecule Förster resonance energy transfer to access the conformational landscape of prothrombin in solution and uncover structural features of functional significance that extend recent x-ray crystallographic analysis. Prothrombin exists in equilibrium between two alternative conformations, open and closed. The closed conformation predominates (70%) and features an unanticipated intramolecular collapse of Tyr(93) in kringle-1 onto Trp(547) in the protease domain that obliterates access to the active site and protects the zymogen from autoproteolytic conversion to thrombin. The open conformation (30%) is more susceptible to chymotrypsin digestion and autoactivation, and features a shape consistent with recent x-ray crystal structures. Small angle x-ray scattering measurements of prothrombin wild type stabilized 70% in the closed conformation and of the mutant Y93A stabilized 80% in the open conformation directly document two envelopes that differ 50 Å in length. These findings reveal important new details on the conformational plasticity of prothrombin in solution and the drastic structural difference between its alternative conformations. Prothrombin uses the intramolecular collapse of kringle-1 onto the active site in the closed form to prevent autoactivation. The open-closed equilibrium also defines a new structural framework for the mechanism of activation of prothrombin by prothrombinase.

Keywords: enzyme kinetics; prothrombin; single-molecule biophysics; structure-function; thrombin.

FIGURE 1.

Prothrombin structure and smFRET. a, high resolution structure of prothrombin devoid of residues 154–167 in Lnk2 (9) (PDB ID 5EDM) showing the overall arrangement of the Gla domain (GD, marine), kringle-1 (K1, red), Lnk2 (wheat), kringle-2 (K2, green), and protease domain comprising the A chain (Ac, orange) and catalytic B chain (Bc, yellow). Ser residues mutated to Cys for conjugation with the thiol-reactive dyes AF555 and AF647 used in smFRET measurements are indicated by purple spheres and labeled. The four FRET couples 34/101, 101/478, 120/478, and 210/478 used in the study (see also panel b) are indicated by dotted lines. b, schematic representation of the modular assembly of prothrombin with the Gla domain (Gla), two kringles (K1 and K2), and protease domain (PD) containing the A and B chains connected by a disulfide bond. Three intervening linkers connect the Gla domain to kringle-1, the two kringles (Lnk2), and kringle-2 to the protease domain. Prothrombin has 24 Cys residues paired into 12 disulfide bonds. FRET couples are listed with their respective domains and the products of digestion of each construct with thrombin (fIIa). Pre-1, prethrombin-1; Pre-2, prethrombin-2. c, incorporation of the probes was checked by limited proteolysis with thrombin. After the addition of 10 μl of loading buffer, proteins were loaded into a gradient 4–12% polyacrylamide gel in the presence of SDS and visualized by Coomassie Brilliant Blue R-250 (black and white) or fluorescence intensity by exciting donor at 532 nm (red panel) and acceptor at 640 nm (blue panel). Prothrombin (proT) wild type shows no detectable fluorescence after being treated under the same conditions. Thrombin cleaves prothrombin at Arg¹⁵⁵ and generates prethrombin-1 and the Gla domain/kringle-1 pair containing residues 101 and 120. A second cleavage at Arg²⁸⁴ produces prethrombin-2 and kringle-2 containing residue 210. The band corresponding to prethrombin-2 appears in the 101/478, 120/478, and 210/478 couples because residue 478 is in the B chain of the protease domain.

FIGURE 2.

Activation of prothrombin constructs by prothrombinase. a–d, all prothrombin mutants labeled for smFRET measurements (filled red circles) are activated by prothrombinase at a rate similar to that of wild type (black circles) or of the unlabeled constructs (open red circles). Measurements were carried out by a continuous assay of substrate hydrolysis (9) using 1 μm prothrombin, 1 pm factor Xa, 20 μm phospholipids, 10 nm cofactor Va, and the chromogenic substrate H-d-Phe-Pro-Phe-p-nitroanilide under experimental conditions: 150 mm NaCl, 5 mm CaCl₂, 0.1% PEG 8000, 20 mm Tris, pH 7.4, at 25 °C. Abs, absorbance. e–h, SDS-PAGE analysis of the conversion of prothrombin (proT, 1.4 μm) to thrombin by prothrombinase (0.2 nm factor Xa, 20 μm phospholipids, 10 nm cofactor Va), that occurs with similar efficiency for all constructs within 180 min. The lack of B chain in the 34/101 construct (e) confirms selective incorporation of the probes in the Gla domain/kringle-1 pair.

FIGURE 3.

smFRET measurements of prothrombin in solution. Shown are histograms of the four FRET couples probing the conformation of prothrombin in solution. The bottom section of the top graph of each construct depicts the stoichiometry, S, versus FRET efficiency for each diffusing molecule that contains both AF555 and AF647 fluorophores. The upper section shows the one-dimensional efficiency histogram of the molecules in the bottom section. Populations were fit to a single (34/101 and 210/478 FRET couples) or double (101/478 and 120/478 FRET couples) Gaussian distribution (red lines). The percentage of each population is indicated. The bottom graphs depict the results of incubation with factor Xa (fXa) or thrombin (fIIa) for 3 h at room temperature. a–d, proteolytic digestion by factor Xa or thrombin minimally affects the 34/101 couple (a), but abrogates the FRET signal for the 101/478 and 120/478 couples (b and c), as expected. The FRET signal for the 210/478 couple (d) disappears upon digestion with factor Xa due to cleavage at Arg²⁷¹. Cleavage at Arg³²⁰ by factor Xa does not separate the A and B chains that remain connected through the disulfide bond between Cys²⁹³ and Cys⁴³⁹. Cleavage at Arg²⁸⁴ by thrombin drastically reduces the signal, as evidenced by the lower counts, but it is not as efficient as the one at R155.

FIGURE 4.

Binding of the active site inhibitor argatroban monitored by smFRET. a, efficiency histograms of the 120/478 couple as a function of argatroban concentration (0–120 μm). Vertical dashed lines indicate the mean efficiency value of free (E = 0.62) and bound (E = 0.27) forms that dominate in the absence or nearly saturating (120 μm) concentrations of argatroban. Histograms were fitted to a double Gaussian distribution (red lines) with well defined single components (black dotted lines). b, the individual areas of the low (bound) and high (free) FRET populations calculated at each concentration of argatroban, normalized by the total change observed upon saturation, show a hyperbolic dependence on the inhibitor concentration. Values of K_d obtained from independent fit of the two curves are 42 ± 5 μm (open circles) and 45 ± 5 μm (closed circles). Data points are averages of triplicate measurements with standard errors within 3%.

FIGURE 5.

The Tyr⁹³–Trp⁵⁴⁷ intramolecular interaction stabilizes the closed form of prothrombin. a, a crystal packing interaction present in all current structures of prothrombin brings Tyr⁹³ from kringle-1 (cyan sticks) in contact with Trp⁵⁴⁷ in the active site of a symmetry-related molecule (yellow sticks). The side chain of Trp⁵⁴⁷ is also stabilized by Trp⁴⁶⁸ in the neighbor autolysis loop of the protease domain. The Tyr⁹³-Trp⁵⁴⁷ interaction occurs in solution within the same prothrombin molecule and occludes the active site in the closed form. b, mutation of Tyr⁹³ or Trp⁵⁴⁷ destabilizes the high FRET closed form and shifts the pre-existing equilibrium of prothrombin in favor of the low FRET open form. c, the conformational difference between open and closed forms is directly documented by proteolytic digestion with chymotrypsin, which readily cleaves the Y93A and W547A mutants within 60 min, but leaves the wild type intact over the same time frame (band at 72 kDa in the acrylamide gel). N-terminal sequencing of the fragments confirms cleavage at Trp⁴⁶⁸ in the mutants (A: ¹ANTLF⁵; B: ⁴⁶⁹TANVG⁴⁷³). This residue is inaccessible to proteolysis in the wild type due to interaction with Trp⁵⁴⁷ (a). d–g, the open and closed forms of prothrombin also differ in their propensity to autoactivate to thrombin (d–f), with (+, red bar) and without (−, grey bar) histone H4 (g), as monitored by SDS-PAGE. The mutant Y93A autoactivates significantly faster than wild type, especially in the presence of histone H4. Replacement of the active site Ser⁵²⁵ with Ala serves as control. Cleavage at Trp¹⁵⁵ is confirmed by the appearance of an intense band that migrates at ∼50 kDa corresponding to prethrombin-1. Errors in panel g are within 2%.

FIGURE 6.

Closed and open conformations of prothrombin revealed by SAXS. a and b, scattering profiles (a) and pair distance distribution functions (b) for prothrombin wild type (orange) and mutant Y93A (blue). c and d, ab initio envelopes calculated from scattering profiles for wild type (c, orange mesh) and mutant Y93A (d, blue mesh) superimposed on an atomic model of the closed conformation (c) or the crystal structure of prothrombin lacking residues 154–167 in Lnk2 (PDB ID 5EDM) (9) (d). The envelope of wild type spans 120 Å and is 50 Å shorter than that of the mutant Y93A. The arrangement of domains in the structure 5EDM is consistent with the elongated SAXS envelope, whose extra volume can be accounted for by the missing portion of Lnk2 (residues 154–167) and the more compact conformation of the Gla domain (GD, marine) in the presence of Mg²⁺ used in the crystallization buffer. Note how transition from the closed to open conformation separates Tyr⁹³ and Trp⁵⁴⁷ (magenta). Wheat, Lnk2; red, kringle-1 (K1); green, kringle-2 (K2); orange, A chain (Ac); yellow, B chain (Bc).