The clotting factor prothrombin exists in equilibrium between closed and open conformations, but the physiological role of these forms remains unclear. As for other allosteric proteins, elucidation of the linkage between molecular transitions and function is facilitated by reagents stabilized in each of the alternative conformations. The open form of prothrombin has been characterized structurally, but little is known about the architecture of the closed form that predominates in solution under physiological conditions. Using X-ray crystallography and single-molecule FRET, we characterize a prothrombin construct locked in the closed conformation through an engineered disulfide bond. The construct: (i) provides structural validation of the intramolecular collapse of kringle-1 onto the protease domain reported recently; (ii) documents the critical role of the linker connecting kringle-1 to kringle-2 in stabilizing the closed form; and (iii) reveals novel mechanisms to shift the equilibrium toward the open conformation. Together with functional studies, our findings define the role of closed and open conformations in the conversion of prothrombin to thrombin and establish a molecular framework for prothrombin activation that rationalizes existing phenotypes associated with prothrombin mutations and points to new strategies for therapeutic intervention.

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. Key biological processes such as blood coagulation and immune response depend on trypsin-like proteases generated from cascades of sequential zymogen activation. Factors involved in these cascades share common ancestry (1) and a modular assembly where the protease domain is coupled to auxiliary components that regulate the proteolytic conversion of zymogen to active enzyme (2). Resolving the spatial organization of these zymogens has long posed a challenge for structural biology. Successes have been few but highly significant (3-7). 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. The zymogen prothrombin offers a biologically relevant example as the key player of the coagulation cascade and one of the most abundant proteins circulating in the blood (8).The modular assembly of prothrombin comprises the Gla domain (residues 1-46), kringle-1 (residues 65-143), kringle-2 (residues 170 -248), and the protease domain (residues 285-579) connected by three intervening linkers (see Fig. 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 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. 1a). Such plasticity has physiological significance: once prothrombin is anchored to the membrane via its Gla domain, Lnk2 pivots the C-terminal kringle-2/protease domain end over the N-terminal Gla domain/kringle-1 end and regulates how the sites of cleavage at Arg 271 and Arg 320 are presented to prothrombinase to promote conversion to the mature enzyme thrombin (9). The plasticity of prothrombin supported by recent crystal structures is compelling, but requires validation from independent measurements in solution where the conformational landscape of the protein is accessible and unconstrained by crystal packing. Similar considerations apply quite generally to other zymogens with modular assembly involved in blood coagulation, immune response, and fibrinolysis. Single molecule Förster resonance energy transfer (smFRET) 3 is an essential tool to probe molecular interactions, folding pathways, and conformational changes of freely diffusing single molecules in solution (11)(12)(13)(14)(15). Advancements in experimental setup and data analysis ensure greater insights * This wo...

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