OC 07.5 - A 3.3Å cryo-EM structure of an engineered high-affinity human prothrombinase complex

Background: Prothrombin is activated by the prothrombinase complex, composed of factor (f) Xa and fVa on membranes. The membrane-dependence of assembly complicates structural studies. We created a high-affinity, membrane-independent prothrombinase by mutating fXa at 17 positions, and have solved its structure to 3.3Å resolution.

Aims: Although a structure of prothrombinase was recently reported at 4.1Å resolution (7TPP), the map contains no feature expected for that resolution and nothing interpretable as side chains. Indeed, none of the reported interactions is supported by the map. Our aim is to obtain a map of prothrombinase of sufficient quality and resolution to determine the intermolecular contacts between fXa and fVa.

Methods: Human fX with 17 mutations was produced in HEK-EBNA cells, activated, and inhibited with EGRCK. B-domainless human fV was produced in BHK-M cells and activated with thrombin. Cryo-EM data were collected on a Titan Krios instrument. 70,154 particles were used to calculate the final map at a resolution of 3.28Å.

Results: As expected from a 3.3Å map, the position of a large proportion of side chains could be interpreted. 4,580 Å2 is buried at the interface, with 9 H-bonds and 23 salt-bridges. The a2-loop of fVa interacts with the heparin binding site of fXa. N-linked glycans and the inhibitor EGRCK are also seen in the map. The mutations that were made to improve affinity can be verified by the map. None of the contacts observed corresponds to any reported for 7TPP.

Conclusion(s): We have engineered a variant of fXa capable of binding to fVa with high affinity in the absence of membranes, and have solved its structure by cryo-EM. The map is of sufficient quality to observe interactions between side chains at the interface, and provides proof that the acidic a2-loop protrudes out and away from fVa to interact with the basic heparin binding site of fXa.