Crystal structure of prothrombin reveals conformational flexibility and mechanism of activation

Abstract

The zymogen prothrombin is composed of fragment 1 containing a Gla domain and kringle-1, fragment 2 containing kringle-2, and a protease domain containing A and B chains. The prothrombinase complex assembled on the surface of platelets converts prothrombin to thrombin by cleaving at Arg-271 and Arg-320. The three-dimensional architecture of prothrombin and the molecular basis of its activation remain elusive. Here we report the first x-ray crystal structure of prothrombin as a Gla-domainless construct carrying an Ala replacement of the catalytic Ser-525. Prothrombin features a conformation 80 Å long, with fragment 1 positioned at a 36° angle relative to the main axis of fragment 2 coaxial to the protease domain. High flexibility of the linker connecting the two kringles suggests multiple arrangements for kringle-1 relative to the rest of the prothrombin molecule. Luminescence resonance energy transfer measurements detect two distinct conformations of prothrombin in solution, in a 3:2 ratio, with the distance between the two kringles either fully extended (54 ± 2 Å) or partially collapsed (≤34 Å) as seen in the crystal structure. A molecular mechanism of prothrombin activation emerges from the structure. Of the two sites of cleavage, Arg-271 is located in a disordered region connecting kringle-2 to the A chain, but Arg-320 is well defined within the activation domain and is not accessible to proteolysis in solution. Burial of Arg-320 prevents prothrombin autoactivation and directs prothrombinase to cleave at Arg-271 first. Reversal of the local electrostatic potential then redirects prothrombinase toward Arg-320, leading to thrombin generation via the prethrombin-2 intermediate.

Keywords: Coagulation factors; Enzyme mutation; Enzyme structure; Structural biology; Thrombin; Zymogen.

FIGURE 1.

Top, schematic representation of prothrombin composed of fragment 1 (residues 1–155), fragment 2 (residues 156–271), and a protease domain (residues 272–579). Fragment-1 contains a Gla domain (residues 1–44) and a kringle (residues 65–143), fragment-2 contains a second kringle (residues 170–248), and the protease domain contains the A (residues 272–320) and B (residues 321–579) chains. Thrombin is generated by two cleavages at Arg-271 and Arg-320, producing the inactive precursor prethrombin-2 and the active intermediate meizothrombin, respectively. The A and B chain remain covalently attached after activation through the disulfide bond Cys-293–Cys-439 (Cys-1–Cys-122). Cleavage at Arg-284 by thrombin itself reduces the length of the A chain to its final 36 amino acids composition. Bottom, x-ray crystal structure of Gla-domainless prothrombin with kringle-1 (red) positioned at an angle of 36° relative to kringle 2 (green) that is coaxial to the protease domain (B chain in yellow and A chain in orange). The active site region is indicated by a circle, and the termini for each domain visible in the orientation are noted. Of the two sites of cleavage, Arg-320 (Arg-15) in the activation domain is visible, but Arg-271 is part of a 20-residue segment missing in the electron density map because of disorder.

FIGURE 2.

a, representative extra electron density detected for fragment 1 after molecular replacement and before refinement. The extra electron density shown as 2F_o − F_c map contoured at 1 σ enables assignment of residues of fragment 1 of Gla-domainless prothrombin with confidence. Shown as sticks are residues Ser-91–His-104 of kringle-1 with the distinguishing Pro-94 that is the single Pro in the cis conformation in the entire kringle, as first identified in the high resolution structure of bovine kringle-1 (6). These residues refer to the structure after refinement, deposited in the Protein Data Bank as entry 4HZH, and show the quality of the model built on the extra electron density detected before refinement after initial molecular replacement. b, overlay of kringle-1 (red) and kringle-2 (green) of Gla-domainless prothrombin reveals the basic similarity between the two domains. Because of the absence of Cys-65 in the electron density map, Cys-143 is unpaired, and only two disulfide bonds (1 = Cys-86–Cys-125, 2 = Cys-114–Cys-138) are detected in kringle-1. All three disulfide bonds (3 = Cys-170–Cys-248, 4 = Cys-191–Cys-231, 5 = Cys-219–Cys-243) are detected for kringle-2. The two kringles align with an r.m.s.d. = 1.2 Å, and the most notable differences (noted by arrows) are a helical segment in kringle-2 replaced by an unstructured coil (residues 92–106) in kringle-1 (see also panel a) and residues 130–134 of kringle-1 shifted relative to the homologous region in kringle-2 by ∼3 Å.

FIGURE 3.

a and b, semilog plot of lifetime data for the LRET donor-acceptor pair conjugated to residues 101 in kringle-1 and 210 in kringle-2 of the full-length prothrombin mutant S101C/S210C. The donor quenching (a, green circles) and acceptor sensitization (b, green circles) both obey the same triple-exponential decay comprising a short lifetime of 33.8 ± 0.5 μs with almost complete (94%) energy transfer and a population of 40% and a long lifetime of 272 ± 4 μs with 55% energy transfer and a population of 60%. The third, slowest lifetime (606 ± 9 μs) is identical to the decay of the donor-only control and free Eu³⁺ chelate (gray circles). The donor-acceptor distances associated with the slow (272 ± 4 μs) and fast (33.8 ± 0.5 μs) decays derived from the Förster equation (see “Experimental Procedures”) are 54 ± 2 and ≤34 Å, respectively. c, molecular dynamics simulations of the prothrombin conformation shown as the Cα-Cα distance between Ser-101 in kringle-1 and Ser-210 in kringle-2 as a function of time, over a 24-ns trajectory. Horizontal red lines depict the values of 54 and 34 Å determined by LRET measurements using probes attached to residues 101 and 210 mutated to Cys. d, overlay of the starting (with domains colored as in Fig. 1) and ending (cyan) prothrombin structures from the molecular dynamics simulation (see panel c). The starting structure is the model of prothrombin bound to prothrombinase (14), and the ending structure is the average of the last 5 ns of the simulation. Residues Ser-101 and Ser-210 are rendered as sticks with Cα-Cα distances indicated in Å.

FIGURE 4.

a and b, x-ray crystal structure of Gla-domainless prothrombin in surface/ribbon representation oriented with active site region (white circle) in the front (a) or rotated 180° (b) showing the bent conformation of the zymogen. Epitopes for factor Xa (yellow) and cofactor Va (red) identified by functional studies are mapped on the surface. The site of cleavage at Arg-271 is in a disordered region, but Arg-320 (Arg-15) is indicated. c and d, electrostatic potential surface of Gla-domainless prothrombin revealing the properties of the epitopes for factor Xa (yellow oval) and cofactor Va (green oval). The surface appears to be predominantly negative, especially in the region connecting kringle-2 to the protease domain housing the epitope for factor Xa binding. The epitope for cofactor Va recognition involves exosite I in the protease domain and is positively charged. The epitope for factor Xa on the C-terminal of the B chain is covered by the acidic residues connecting kringle-2 to the A chain and the junction between the two kringles. The electrostatic properties of this epitope change drastically upon cleavage at Arg-271 that generates prethrombin-2. e and f, cleavage at Arg-271 generates prethrombin-2 by removing the acidic linker between kringle-2 and the A chain and exposes the positively charged surface of exosite II on the protease domain. On the other hand, cleavage at Arg-271 has no effect on the epitope for cofactor Va recognition.

FIGURE 5.

a, stereo image of the active site of Gla-domainless prothrombin. The absence of a new N terminus at residue Ile-321 (Ile-16) causes a shift in the position of Asp-524 (Asp-194) resulting in disruption of the oxyanion hole (arrow) defined by the tight β-turn between the backbone N atoms of the catalytic Ser-525 (Ser-195) and Gly-523 (Gly-193). The side chain of Asp-519 (Asp-189) in the primary specificity pocket flips almost 7 Å from the position occupied in thrombin. The 547–549 (215–217) segment collapses in the active site, a signature of the inactive E* form. Trp-468 (Trp-148) in the autolysis loop was removed for clarity. b, stereo image of the activation domain of Gla-domainless prothrombin. Arg-320 (Arg-15) is clearly visible in the electron density map with an intact Arg-320–Ile-321 (Arg-15–Ile-16) peptide bond. Ionic interaction with Glu-323 (Glu-18) reduces solvent exposure for proteolytic attack of Arg-320 (Arg-15) by 32%. Electron density 2F_o − F_c maps are contoured at 1 σ.

FIGURE 6.

a and b, cleavage of Gla-domainless prothrombin (GD-ProT) mutants S525A and E311A/D318A/E323A/S525A (EDES) by thrombin, under experimental conditions of 20 mm Tris, 200 mm NaCl, 2 mm CaCl₂, pH 7.4 at 25 °C. The chemical identity of each band in the gels, labeled with the same color as the kinetic traces (c and d), was assigned by N-terminal sequencing. c and d, both mutants are cleaved with similar kinetic rates at Arg-155 to generate prethrombin-1 (Pre1) and Arg-284 to generate prethrombin-2 (Pre2), proving that these sites have similar solvent exposure. On the other hand, Arg-320 (Arg-15) is cleaved only in the mutant E311A/D318A/E323A/S525A (d), suggesting that this Arg residue is trapped by residues of the Glu-311/Asp-318/Glu-323 (Glu-14e/Asp-14l/Glu-18) anionic cage as seen in prethrombin-2 (16). Thr, thrombin.