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 ¼ 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 þ 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.X-ray crystallography | blood coagulation | E* form A ctivation of trypsin-like proteases requires proteolytic processing of an inactive zymogen precursor (1) that occurs at the identical position in all known members of the family, i.e., between residues 15 and 16 (chymotrypsin numbering). The nascent N terminus induces formation of an ion pair with the highly conserved D194 that organizes both the oxyanion hole and primary specificity pocket for substrate binding and catalysis (2, 3). Through this mechanism, the zymogen irreversibly converts into the mature enzyme and affords regulation of catalytic activity. Importance of this paradigm is particularly evident in the activation, progression, and amplification of enzyme cascades, where each component acts as a substrate in the zymogen form in one step and as an enzyme in the subsequent step (4). Yet, recent findings suggest that this paradigm needs revision to account for the conformational plasticity of the protein acting as an enzyme or substrate (5, 6).In the blood coagulation cascade, the protease thrombin is generated from the zymogen prothrombin by the prothrombinase complex, composed of factors Va and Xa, phospholipid membranes, and Ca 2þ (7). Prothrombin is composed of a Gla domain, two kringle domains and the trypsin-like catalytic domain (Fig. 1). The prothrombinase complex converts prothrombin to thrombin along two pathways by cleaving sequentially at R271 and R320 (prothrombin numbering). Initial cleavage at R320 (R15 in the chymotrypsin numbering) between the A and B chains is the preferred pathway under phy...

The linker connecting the two kringles plays a key role in prothrombin activation

“…The occluded conformation of the active site of ProTΔ146-167 is caused mainly by the repositioning of W468 (W148) and not by the collapse of W547 (W215) observed in the structures of other inactive thrombin precursors such as GD-ProT (15), prethrombin-1 (17), and prethrombin-2 (25). The width of the active site of ProTΔ146-167 is similar to that reported for the zymogen of complement factor C1r (26) and trypsinogen (27) and falls toward the lower end of the distribution of trypsin-like zymogens (28) that ranges from 8.1 Å in the zymogen of complement factor D (29) to 14.6 Å in prethrombin-1 (17). Another critical Cα-Cα distance between the catalytic S525 (S195) and D519 (D189) measures the depth of the active site pocket and increases slightly from 14.4 Å in GD-ProT to 15.6 Å in ProTΔ146-167 (Fig.…”

“…Complete disorder was detected in Lnk1, residues 144-168 of Lnk2, and residues 255-273 of Lnk3 containing the site of cleavage at R271. Disorder of Lnk3 was already documented in previous structures of meizothrombin desF1 (16) and prethrombin-1 (17), but the flexibility of Lnk1 and Lnk2 could not be anticipated before the structure of GD-ProT became available. Luminescence resonance energy transfer measurements of prothrombin in solution with probes attached to kringle-1 and kringle-2 have shown that Lnk2 assumes a range of conformations and functions like a flexible spring connecting the kringle-2/protease domain pair on the C-terminal side to the Gla domain/kringle-1 pair on the N-terminal side (15).…”

Crystal structure of the prothrombinase complex from the venom of Pseudonaja textilis

“…The crystal structure of the N-terminal portion, composed of the Gla and kringle 1 domains (together known as F1), revealed a tight association between the 2 domains into a single rigid unit. 35 The C-terminal portion of prothrombin is composed of the second kringle domain (K2) and the catalytic domain, which also associate into a rigid unit due to the tight interaction of K2 with the catalytic domain (together known as prethrombin-1 [Pre1] 36 ). F1 and Pre1 are connected by a 26-residue unstructured linker, suggesting that each rigid domain binds independently to prothrombinase.…”

“…42 The structural features of Pre1 and meizothrombin, coupled with the identification of the putative substrate binding site on prothrombinase help to rationalize the ratcheting mechanism. The structures of Pre1 and meizothrombin 36,43 show that Pre1 undergoes a large conformational rearrangement when converted to meizothrombin. The conformational change involves the ordering of the catalytic domain and a repositioning of the K2 domain.…”

The Fragment 1 Region of Prothrombin Facilitates the Favored Binding of Fragment 12 to Zymogen and Enforces Zymogen-like Character in the Proteinase

“…Although the foregoing provides a reasonable explanation for our findings, it cannot be readily reconciled with all structural information. The F2 domain in the prethrombin 1 structure (lacking the F1 domain) readily superimposes on the F2 domain in the three other structures of prothrombin retaining covalent linkage with K1 and the Gla domain (11,12,31). The pairwise root mean square deviations for these superimpositions ranged from 0.29 -0.78 Å (mean 0.53 Å), suggesting the lack of a major effect of K1 or cleavage at Arg 155 on the folding of K2.…”