Cryo-EM structures of coagulation factors

Abstract

A State of the Art lecture titled "Cryo-EM structures of coagulation factors" was presented at the ISTH Congress in 2022. Cryogenic electron microscopy (cryo-EM) is a revolutionary technique capable of solving the structure of high molecular weight proteins and their complexes, unlike nuclear magnetic resonance (NMR), and under conditions not biased by crystal contacts, unlike X-ray crystallography. These features are particularly relevant to the analysis of coagulation factors that are too big for NMR and often recalcitrant to X-ray investigation. Using cryo-EM, we have solved the structures of coagulation factors V and Va, prothrombinase on nanodiscs, and the prothrombin-prothrombinase complex. These structures have advanced basic knowledge in the field of thrombosis and hemostasis, especially on the function of factor V and the molecular mechanism for prothrombin activation, and set the stage for exciting new lines of investigation. Finally, we summarize relevant new data on this topic presented during the 2022 ISTH Congress.

Keywords: blood coagulation; factor V; factor Va; prothrombin; vitamin K‐dependent clotting factors.

FIGURE 1

Cryo‐EM structure of fV. (A) Schematic representation of the A1‐A2‐B‐A3‐C1‐C2 domain organization of human fV (2196 residues total) and its B domain containing the basic (BR) and acidic (AR) regions that interact to keep fV inactive. The short hydrophobic patch is unmasked in the splice variant fV short and contributes to TFPIα binding, along with the acidic region. (B) The protein is rendered in surface representation with the constitutive domains colored in wheat (A1), pale green (A2), light blue (B), pale yellow (A3), light pink (C1), and pale cyan (C2). The structure of fV (7KVE ¹⁹ ) was solved at 3.3 Å resolution and features the C domains aligned “edge‐to‐edge” into a platform involved in membrane binding and upon which the A domains rest side by side. The A1‐A2‐A3‐C1‐C2 domain assembly is resolved in its entirety. The sites of thrombin activation at R709 and R1545 (magenta) are clearly visible in the A2 and B domains and exposed to solvent for proteolytic attack. The sites of APC cleavage at R306 and R506 (red) are 75% buried. Also shown are the gate (blue) and the lid (orange) that play an important role in prothrombin activation. The B domain is very dynamic and only a total of 14 residues are resolved (red circles) in the connection to the A2 and A3 domains.

FIGURE 2

Cryo‐EM structure of fVa free and bound to fXa. (A) The protein is rendered in surface representation with the constitutive domains colored as in Figure 1B. Overall, the arrangement of the A1‐A2‐A3‐C1‐C2 domains is like that of fV. The structure of fVa (7KXY ¹⁹ ) was solved at 4.4 Å resolution and is more disordered than that of fV, with fewer (1181 of 1360 total) residues resolved in the A1 (294 of 316 total), A2 (294 of 393 total), A3 (295 of 332 total), and C2 (139 of 160 total) domains. The disorder in fVa removes all information about the gate and lid (dotted circle). (B) The structure of prothrombinase was solved on nanodiscs (7TPQ ²⁰ ) at 5.3 Å resolution and shows fVa and fXa in surface representation, with fVa colored as in Figure 1 and fXa in red. The architecture of the complex is solved in almost its entirety (1742 residues of 1752), except for the N‐terminal ¹⁵⁴⁶SNNGNRRNYY¹⁵⁵⁵ sequence of the A3 domain immediately downstream of the site of thrombin activation at R1545. The overall arrangement of fVa is like that of the free form (A) but the gate and lid are fully resolved and change their conformation relative to fV (Figure 1B). The architecture of fXa is also fully resolved for the first time and shows a curved conformation, enhanced by a 90° kink at the EGF1‐Gla domain junction, that positions the active site about 60 Å over the plane of the nanodiscs. The enzyme aligns along the A2, A3, and C1 domains of fVa, with most of the contacts between the A2 domain of fVa and the protease domain (PD) of fXa (Table 1).

FIGURE 3

Prothrombin activation pathways. Schematic representation of prothrombin activation by the prothrombinase complex. The conversion to thrombin entails cleavage at R271 in Lnk3 and R320 in the A chain, along two alternative pathways generating the intermediates prethrombin‐2 (cleavage at R271 first) or meizothrombin (cleavage at R320 first). An additional autoproteolytic cleavage at R284 produces thrombin. The presence of fVa, in vitro, promotes activation along the meizothrombin pathway. In the absence of fVa, activation proceeds along the prethrombin‐2 pathway or through cleavage at R155 to produce prethrombin‐1.

FIGURE 4

Cryo‐EM structure of the prothrombin‐prothrombinase complex. (A) The structure (7TPP ²⁰ ) was solved at 4.1 Å resolution and shows prothrombinase in the same arrangement found on nanodiscs (Figure 2B) and prothrombin (yellow) aligning along the C2, A1, and A2 domains of fVa in a curved conformation similar to the closed form identified crystallographically. The architecture of the complex is solved in almost its entirety (2307 residues of 2331). Remarkably, prothrombinase engages prothrombin only through the protease domain (PD) that binds to the gate of fVa (covered) and the active site region of fXa. The Gla domain of prothrombin aligns with the Gla domain of fXa and the C1 and C2 domains of fVa to define a membrane binding module but remains 100 Å separated from prothrombinase. The site of cleavage at R320 penetrates the active site of fXa (see Figure 5). The structure depicts prothrombinase in the process of cleaving prothrombin at R320 to initiate activation along the meizothrombin pathway. (B) Representative 2D class averages of the prothrombin‐prothrombinase complex (bottom) and images of fVa (top) document the distribution of free and bound particles obtained under the conditions used for cryo‐EM structure determination.

FIGURE 5

Mechanism of prothrombin activation along the meizothrombin pathway. Top view of the prothrombin‐prothrombinase complex showing fVa in surface representation (green) and the protease domains of fXa (red) and prothrombin (yellow) as cartoons. The lid drops >7 Å on the protease domain of fXa (blue arrow) and fixes the orientation of the active site region to optimize interaction with prothrombin. The protease domain of fXa makes extensive interactions (Table 1) with the A2 domain of fVa, especially through the Na⁺ binding site, the 350 segment and the C‐terminal helix. The Ca⁺ binding site is on the opposite side of the fVa‐fXa interface. Residues of the catalytic triad are in red and D373 in the primary specificity pocket is shown in green. The sites of activation of prothrombin, R320 and R271, are shown in green and are widely separated. Important regions of the zymogen are Lnk3 (gray) that supports R271 and continues on to the A chain (cyan) hosting R320 ready to engage D373 of fXa. The B chain (yellow) contains exosite I (magenta) that makes no contacts with prothrombinase. The only segment of prothrombin contacting prothrombinase is the A chain (cyan), in agreement with clinical data on naturally occurring mutations associated with severe bleeding. The mechanism of prothrombin activation involves a drop of the lid on fXa to curve its conformation and fix orientation of the active site and a conformational change of the gate that sequesters R271 against D697 on one side and provides a surface on the other side on which the A chain slides down to present R320 to the active site of fXa.