Structural Architecture of Prothrombin in Solution Revealed by Single Molecule Spectroscopy

Abstract

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...

Non-canonical proteolytic activation of human prothrombin by subtilisin from Bacillus subtilis may shift the procoagulant–anticoagulant equilibrium toward thrombosis

“…Notably, the subtilisin cleavage sites in ProT perfectly match with the regions of highest segmental flexibility of the zymogen, as deduced from the B-factor plot of the crystallographic structure in the solid state (4) and single-molecule FRET studies in solution ( Fig. 3C) (31). In particular, CS-1 and CS-2 are embedded in the highly dynamic inter-domain linker regions, whereas CS-3 is in the highly flexible 148-loop of the Pre2 domain ( Fig.…”

“…3) (4,32), corresponding to the autolysis loop in the mature ␣T (33). Of note, the structure of ProT we used here is that of the zymogen deletion mutant, ProT⌬(154 -167), solved at 2.2-Å resolution and lacking 14 amino acids in the linker-2 region, while retaining the conformational properties of the wild-type ProT in the extended conformation (29,31). A key aspect that emerges by comparing the time-course increase of hydrolytic activity at 37°C (Fig.…”

“…difference can be accounted for by the following: (i) the lower number of subtilisin cleavages sites present on Pre2 structure compared with ProT; (ii) the lack of glycosylation at Asn-60g that in the natural glycosylated ProT is expected protect the zymogen from subtilisin cleavage (30); and (iii) the possible shielding of the 148-loop operated by the N-terminal F1 region, which in the solution structure of ProT has been proposed to fold back on the Pre2 domain (31).…”

X-ray and solution structures of human beta-2 glycoprotein I reveal a new mechanism of autoantibody recognition

“…The high ionic strength used in the crystallization buffers may destabilize hydrogen bonds and favor hydrophobic interactions thus forcing the protein to assume a non-native conformation(2). To address this concern, we applied smFRET to β 2 GPI(37, 41, 42). By recording the energy that is transferred from an excited molecule (Donor) to a second molecule with spectral overlap (Acceptor) at the single molecule level, smFRET measures distances on a nanometer scale thus serving as a molecular ruler.…”

Protein engineering by chemical methods: Incorporation of nonnatural amino acids as a tool for studying protein folding, stability, and function

“…For this reason, molecules able to inhibit αT activity are the focus of intensive pharmacological research. αT displays a unique surface electrostatic potential (Figure ), generated by the asymmetry of charge distribution, where a negatively charged active‐site region is flanked by two positive patches opposite to the active site, that is, exosites 1 and 2, and responsible for physiological substrates binding . In hemostasis, αT exerts either procoagulant or anticoagulant functions .…”