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

Abstract

The zymogen prothrombin is proteolytically converted by factor Xa to the active protease thrombin in a reaction that is accelerated >3,000-fold by cofactor Va. This physiologically important effect is paradigmatic of analogous cofactor-dependent reactions in the coagulation and complement cascades, but its structural determinants remain poorly understood. Prothrombin has three linkers connecting the N-terminal Gla domain to kringle-1 (Lnk1), the two kringles (Lnk2), and kringle-2 to the C-terminal protease domain (Lnk3). Recent developments indicate that the linkers, and particularly Lnk2, confer on the zymogen significant flexibility in solution and enable prothrombin to sample alternative conformations. The role of this flexibility in the context of prothrombin activation was tested with several deletions. Removal of Lnk2 in almost its entirety (ProTΔ146-167) drastically reduces the enhancement of thrombin generation by cofactor Va from >3,000-fold to 60-fold because of a significant increase in the rate of activation in the absence of cofactor. Deletion of Lnk2 mimics the action of cofactor Va and offers insights into how prothrombin is activated at the molecular level. The crystal structure of ProTΔ146-167 reveals a contorted architecture where the domains are not vertically stacked, kringle-1 comes within 9 Å of the protease domain, and the Gladomain primed for membrane binding comes in contact with kringle-2. These findings broaden our molecular understanding of a key reaction of the blood coagulation cascade where cofactor Va enhances activation of prothrombin by factor Xa by compressing Lnk2 and morphing prothrombin into a conformation similar to the structure of ProTΔ146-167.clotting factor | zymogen activation | structural biology T he clotting factor prothrombin is proteolytically converted by factor Xa to the active protease thrombin by cleavage at two sites, R271 and R320, along two mutually exclusive pathways producing the intermediates prethrombin-2 (cleavage at R271 first) or meizothrombin (cleavage at R320 first). The reaction is greatly accelerated through a drastic increase in k cat when catalyzed by the prothrombinase complex composed of factor Xa, cofactor Va, Ca 2+ , and phospholipids (1, 2). Both the rate enhancement and pathway of activation are under the control of cofactor Va and phospholipids (3, 4), although the cofactor has the greatest effect in enhancing k cat . The molecular determinants of this physiologically important effect are of substantial mechanistic interest because they bear on the cofactor-dependent activation of zymogens in many proteolytic cascades (5, 6).Prothrombin is composed of fragment 1 (residues 1-155), fragment 2 (residues 156-271), and a protease domain (residues 272-579). Fragment 1 contains the Gla domain (residues 1-46) and kringle-1 (residues 65-143), fragment 2 contains kringle-2 (residues 170-248), and the protease domain contains the A chain (residues 272-320) and the catalytic B chain (residues 321-579). Three linkers-to be referred to as Lnk1, Lnk2, ...

Structural Architecture of Prothrombin in Solution Revealed by Single Molecule Spectroscopy

“…3a) with a signal retained upon incubation with thrombin or factor Xa, as expected. The results are consistent with all current crystal structures of prothrombin (7,9,10). Assuming a Förster distance R o ϭ 51 Å for the AF555/AF647 FRET couple, the calculated interprobe distances of 34/101 and 210/478 are 34 Ϯ 2 and 58 Ϯ 4 Å, respectively.…”

“…3d). The signal is retained upon cleavage by thrombin at Arg 155 (7) in Lnk2, but disappears upon cleavage by factor Xa at Arg 271 . Likewise, the FRET couple 34/101 below Lnk2 reports a single population (Fig.…”

“…Resolving the spatial organization of these zymogens has long posed a challenge for structural biology. Successes have been few but highly significant (3)(4)(5)(6)(7). In some cases, multiple relative arrangements of individual domains have been detected from x-ray analysis, underscoring both the plasticity of the fold and the need for validation from solution studies where the protein is unconstrained by crystal packing.…”

“…1, a and b). The linker connecting the two kringles (Lnk2) spans residues 144 -169 and is highly flexible (7,9). Crystallization of prothrombin has succeeded only recently and required deletion of the Gla domain (10) or significant portions of the flexible Lnk2 (7,9).…”

“…The linker connecting the two kringles (Lnk2) spans residues 144 -169 and is highly flexible (7,9). Crystallization of prothrombin has succeeded only recently and required deletion of the Gla domain (10) or significant portions of the flexible Lnk2 (7,9). The molecular picture emerging from these structures is that prothrombin is organized as two rigid ends, the N-terminal Gla domain/kringle-1 pair and the C-terminal kringle-2/protease domain pair, capable of different relative arrangements mediated by Lnk2 (see Fig.…”