Crystal structure of prethrombin-1

Abstract

Prothrombin is the zymogen precursor of the clotting enzyme thrombin, which is generated by two sequential cleavages at R271 and R320 by the prothrombinase complex. The structure of prothrombin is currently unknown. Prethrombin-1 differs from prothrombin for the absence of 155 residues in the N-terminal domain and is composed of a single polypeptide chain containing fragment 2 (residues 156-271), A chain (residues 272-320), and B chain (residues 321-579). The X-ray crystal structure of prethrombin-1 solved at 2.2-Å resolution shows an overall conformation significantly different (rmsd = 3.6 Å) from that of its active form meizothrombin desF1 carrying a cleavage at R320. Fragment 2 is rotated around the y axis by 29° and makes only few contacts with the B chain. In the B chain, the oxyanion hole is disrupted due to absence of the I16-D194 ion pair and the Na(+) binding site and adjacent primary specificity pocket are highly perturbed. A remarkable feature of the structure is that the autolysis loop assumes a helical conformation enabling W148 and W215, located 17 Å apart in meizothrombin desF1, to come within 3.3 Å of each other and completely occlude access to the active site. These findings suggest that the zymogen form of thrombin possesses conformational plasticity comparable to that of the mature enzyme and have significant implications for the mechanism of prothrombin activation and the zymogen → protease conversion in trypsin-like proteases.

Fig. 1.

Schematic representation of prothrombin activation. Prothrombin contains a Gla domain, two kringles (K1 and K2), and a protease domain composed of the A and B chains. Cleavage at R320 separates the A and B chains and generates an active protease. Meizothrombin and prethrombin-2 are generated by a single cleavage at R320 or R271, respectively. Cleavage at both sites generates thrombin. The zymogen prethrombin-1 differs from prothrombin for the lack of the Gla domain and first kringle. The active form of prethrombin-1 is meizothrombin desF1.

Fig. 2.

Crystal structure of prethrombin-1 at 2.2-Å resolution (Upper) compared to the structure of meizothrombin desF1 (Lower) (11). Fragment 2 (gold) docking on the B chain (green) and making no contacts with the A chain (red) is moved > 5 Å upward and rotated 29° around the y axis relative to the position seen in the active meizothrombin desF1 bound to PPACK (yellow sticks). Nineteen residues, from E255 to A273, connecting fragment 2 to the A chain in the back of the molecule (Right), are missing in the electron density map, as opposed to 36 residues in meizothrombin desF1. The site of cleavage between the A and B chains at R320 is fully exposed to solvent. In the front view (Left), the perturbed conformation of the B chain of prethrombin-1 is readily apparent. The side chains of Y60a, W60d, L99, W148, and W215 (orange) coalesce and occlude access to the active site.

Fig. 3.

Contacts between the B chain with the A chain and fragment 2 of prethrombin-1. Fragment 2 (gold cartoon and sticks) assumes the expected fold for a kringle domain but makes few contacts (sticks) with the B chain rendered as a surface in wheat (atoms < 4 Å away are in cyan). Details of these contacts are given in the text. Three disulfide bonds involving the Cys pairs 170–248 (A), 219–243 (B), and 191–231 (C) are fully resolved in fragment 2, and so are W194 and W230 that are stacked. The A chain (green cartoon and sticks) decorates the back of the prethrombin-1 molecule (atoms < 4 Å away are in orange) without making contacts with fragment 2. The site of mutation at A284 is clearly visible in the 2F_o-F_c density map (contoured at 1σ, green mesh) and so is the trident formed by F280, F281, and F286 that penetrates a deep crevice of the B chain. Shown are selected residues (sticks) that make contacts with the B chain, along with the terminal residues Q169 and E254 for fragment 2 and T274 for the A chain. Residues of fragment 2 and the A chain are labeled in black and relevant residues on the surface of the B chain are labeled in white.

Fig. 4.

Activation domain and zymogen architecture of the oxyanion hole in prethrombin-1. (A) The segment around the cleavage site at R320 (R15 in the chymotrypsin numbering) defines the activation domain and shows an intact R15–I16 peptide bond and a conformation similar to that of prethrombin-2 (8). The electron density 2F_o-F_c map (green mesh) is contoured at 1σ. (B) Lack of the I16-D194 ion pair forces the side chain of D194 to seek alternative H-bonding partners in the backbone N atoms of W141, G142 and N143. The latter backbone N atom forms a key H bond with the backbone O atom of E192 in the active protease (15). Disruption of this H bond causes the E192-G193 peptide bond to flip (arrow) with resulting disruption of the oxyanion hole formed by the backbone N atoms of G193 and S195. Added perturbation to the architecture of the oxyanion hole comes from the side chain of N143 that H bonds to the backbone N atom of the catalytic S195. The electron density 2F_o-F_c map (green mesh) is contoured at 2σ.

Fig. 5.

Hydrophobic cluster of prethrombin-1. A cluster of hydrophobic/aromatic residues completely occludes access to the active site of prethrombin-1 (gold cartoon and sticks). The cluster is formed by the collapse of W215 and W148 into the active site, with the indole rings moving 8–10 Å away from their positions in prethrombin-2 [cyan, Protein Data Bank (PDB) ID 1HAG] (8) or meizothrombin desF1 (11). Y60a, W60d, L99, W148, and W215 (gold) are in van der Waals interaction (dotted lines with distances). The collapse of W215 into the active site is similar to that observed in the E* form of thrombin (white, PDB ID 3BEI) (–15). Crystal contacts with symmetry related molecules in the lattice are > 4.6 Å away in the region surrounding the hydrophobic cluster. The electron density 2F_o-F_c map (green mesh) is contoured at 2σ.