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. 2016 Jul 19;291(35):18107–18116. doi: 10.1074/jbc.M116.738310

FIGURE 1.

FIGURE 1.

Prothrombin structure and smFRET. a, high resolution structure of prothrombin devoid of residues 154–167 in Lnk2 (9) (PDB ID 5EDM) showing the overall arrangement of the Gla domain (GD, marine), kringle-1 (K1, red), Lnk2 (wheat), kringle-2 (K2, green), and protease domain comprising the A chain (Ac, orange) and catalytic B chain (Bc, yellow). Ser residues mutated to Cys for conjugation with the thiol-reactive dyes AF555 and AF647 used in smFRET measurements are indicated by purple spheres and labeled. The four FRET couples 34/101, 101/478, 120/478, and 210/478 used in the study (see also panel b) are indicated by dotted lines. b, schematic representation of the modular assembly of prothrombin with the Gla domain (Gla), two kringles (K1 and K2), and protease domain (PD) containing the A and B chains connected by a disulfide bond. Three intervening linkers connect the Gla domain to kringle-1, the two kringles (Lnk2), and kringle-2 to the protease domain. Prothrombin has 24 Cys residues paired into 12 disulfide bonds. FRET couples are listed with their respective domains and the products of digestion of each construct with thrombin (fIIa). Pre-1, prethrombin-1; Pre-2, prethrombin-2. c, incorporation of the probes was checked by limited proteolysis with thrombin. After the addition of 10 μl of loading buffer, proteins were loaded into a gradient 4–12% polyacrylamide gel in the presence of SDS and visualized by Coomassie Brilliant Blue R-250 (black and white) or fluorescence intensity by exciting donor at 532 nm (red panel) and acceptor at 640 nm (blue panel). Prothrombin (proT) wild type shows no detectable fluorescence after being treated under the same conditions. Thrombin cleaves prothrombin at Arg155 and generates prethrombin-1 and the Gla domain/kringle-1 pair containing residues 101 and 120. A second cleavage at Arg284 produces prethrombin-2 and kringle-2 containing residue 210. The band corresponding to prethrombin-2 appears in the 101/478, 120/478, and 210/478 couples because residue 478 is in the B chain of the protease domain.