Putative complex of fV short bound to prothrombin and fXa. Complex of prothrombin (yellow) and fXa (red) bound to fV short (colored as in Figure 2) generated by replacing fVa in the prothrombin-prothrombinase complex16 with the cryo-EM structure of fV short, without further optimization. Overall, the structure of fV short accounts for its fVa-like activity1,5,8,9,14 and points to modest conformational changes needed to optimize interaction with prothrombin and fXa. (A,B) Prothrombin aligns along the C2, A1, and A2 domains and makes no contacts with the B domain. The site of cleavage at R320 (not visible) penetrates the active site of fXa to promote activation along the meizothrombin pathway,16 whereas R271 (not visible) and R155 (light blue) remain widely separated from the enzyme. A backbone clash (red oval) involves the segment 260LDEDSDRAIE269 preceding the site of cleavage of prothrombin at R271 and the segment 693DADYDYQNRL702 of the A2 domain that includes the gate (blue). The clash is triggered by the repositioning of the lid (orange) and removes the important interaction between R271 and D697 of the gate (shifted 12 Å) that directs R320 to the active site of fXa.10,16 Removal of the clash would require slight rearrangement of the gate and/or the A chain of prothrombin. (B,C) fXa aligns along the A2, A3, and C1 domains of fV short. Repositioning of the lid removes numerous interactions with fXa observed in the prothrombinase complex16 and exposes the acidic segment 658PDDDEDSYEIFEPP671 (Figure 2A and 4A) for possible new contacts with the enzyme (white oval). The proximity of this segment to the AR generates an extended surface of negative electrostatic potential (supplemental Figure 4) for the engagement of the protease domain of fXa and explains how intramolecular interaction of this region with the BR in fV may keep the cofactor in its inactive state.1,5-7,9,53 In fV short, this region likely engages the basic C-terminal end of TFPIα, leading to compromised fVa-like activity.7 The proximity to R506 also explains the TFPIα difficult interaction with fVLeiden (R506Q)54,55 and its ability to protect R506 from APC cleavage.27 A backbone clash (orange oval) involves the AR segment 1530INSSRDPDNIAAVYLR1545 of fV short with the segment 86RKLCSLDN93 of the EGF2 domain of fXa. Removal of this clash would require a modest (<3 Å) relative rearrangement of the 2 segments. No significant clashes involve fXa and other regions of the B domain, including the I755-L1459 junction at the splice site of our construct (Figure 3A).
