The X-ray Crystal Structure of Full-Length Human Plasminogen

Background: The x-ray structure and mechanism of activation of prothrombin remain elusive. Results: X-ray and solution studies document conformation flexibility of prothrombin. The two sites of cleavage at Arg-271 and Arg-320 have distinct solvent accessibility. Conclusion: Burial of Arg-320 prevents prothrombin autoactivation and directs prothrombinase to cleave at Arg-271 first. Significance: A structure-based mechanism of prothrombin activation emerges.

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Crystal Structure of Prothrombin Reveals Conformational Flexibility and Mechanism of Activation

Pozzi

Chen

Gohara

et al. 2013

“…Protection of the zymogen form from autoproteolytic or proteolytic cleavage is a common theme among proteins with modular assembly. Plasminogen assumes a closed form stabilized by intramolecular interaction of the protease domain with kringles that keeps the zymogen in an activation-resistant conformation (6). Binding of kringles to fibrin clots and cell surface receptors is assumed to induce a transition to an open form that can be cleaved and converted to plasmin by physiological activators.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

Structural Architecture of Prothrombin in Solution Revealed by Single Molecule Spectroscopy

Pozzi

Bystranowska

Zuo

et al. 2016

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...

“…Plasminogen is a 92-kDa zymogen that includes a PNA-apple N-terminal domain, five kringle domains (K1-5), and a serine proteinase catalytic domain (23). The kringle domains mediate interactions with fibrin clots and surface receptors from both eukaryotic and bacterial cells (24,25).…”

mentioning

confidence: 99%

Molecular Interactions of Human Plasminogen with Fibronectin-binding Protein B (FnBPB), a Fibrinogen/Fibronectin-binding Protein from Staphylococcus aureus

Pietrocola

Nobile

Gianotti

et al. 2016

Staphylococcus aureus is a commensal bacterium that has the ability to cause superficial and deep-seated infections. Like several other invasive pathogens, S. aureus can capture plasminogen from the human host where it can be converted to plasmin by host plasminogen activators or by endogenously expressed staphylokinase. This study demonstrates that sortase-anchored cell wall-associated proteins are responsible for capturing the bulk of bound plasminogen. Two cell wall-associated proteins, the fibrinogen-and fibronectin-binding proteins A and B, were found to bind plasminogen, and one of them, FnBPB, was studied in detail. Plasminogen captured on the surface of S. aureusor Lactococcus lactis-expressing FnBPB could be activated to the potent serine protease plasmin by staphylokinase and tissue plasminogen activator. Plasminogen bound to recombinant FnBPB with a K D of 0.532 M as determined by surface plasmon resonance. Plasminogen binding did not to occur by the same mechanism through which FnBPB binds to fibrinogen. Indeed, FnBPB could bind both ligands simultaneously indicating that their binding sites do not overlap. The N3 subdomain of FnBPB contains the full plasminogen-binding site, and this includes, at least in part, two conserved patches of surface-located lysine residues that were recognized by kringle 4 of the host protein.