Putative interaction of prothrombin in the open form with prothrombinase. (A) The complex was obtained by overlaying the structure of prothrombin in the open form (PDB ID 5EDM)³⁰ to the closed form in the cryo-EM structure (Figures 1B and 2). The open form aligns the Gla domain with the homologous domain of fXa and the C1 and C2 domains of fVa. Unfortunately, the entire segment ²⁵⁷GDGLDEDSDRAIEGRTAT²⁷⁴ containing the site of cleavage at R271 was not resolved in the 5EDM structure (B, dotted lines). Residue R320 moves 18 Å upward and clashes with the area of contact between E686 of fVa and K376 of fXa (yellow circle; see also Figure 5). A movement of the entire 672 to 691 region (gray) of the A2 domain would be necessary to accommodate the protease domain of prothrombin in the open form. Exosite-1 of prothrombin (residues ³⁸²RIGKHSRTRYERNIE³⁹⁶, orange) moves closer to but not in contact with fVa. (B-C) Protease domain of prothrombin in the open (B) and closed (C) forms obtained after rotation of panel A 90° clockwise along the y-axis (B) or directly from Figure 2E (C), with fVa, fXa, and the auxiliary domains (EGF1, EGF2, Gla) removed for clarity. Transition from the closed to the open form moves exosite 1 closer to fVa and relocates R320 18 Å upward from the position in the closed form (indicated in panel B by a yellow circle, for reference). The transition also causes a significant clockwise rotation of the entire segment 257 to 274 containing the R271 site (C), not visible in the open form (B, dotted lines), with the Cα-Cα distance between T256 and S275 shrinking from 37 to 16 Å. The rotation would bring R271 closer to the active site of fXa in the open form.