9I2H

A 3.3 angstrom cryo-EM structure of an engineered high-affinity human prothrombinase complex

This is a non-PDB format compatible entry.

Summary for 9I2H

• Entry DOI: 10.2210/pdb9i2h/pdb
• EMDB information: 52585
• Related PRD ID: PRD_000288
• Descriptor: Activated factor Xa heavy chain, SODIUM ION, L-alpha-glutamyl-N-{(1S)-4-{[amino(iminio)methyl]amino}-1-[(1S)-2-chloro-1-hydroxyethyl]butyl}glycinamide, ... (12 entities in total)
• Functional Keywords: enzyme, complex, prothrombinase, blood clotting
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 4
• Total formula weight: 205325.79

Authors

Huntington, J.A.,Faille, A.,Ustok, F.I. (deposition date: 2025-01-20, release date: 2025-11-26)

Primary citation

Ustok, F.I.,Faille, A.,Huntington, J.A.
A 3.3- angstrom cryo-EM structure of an engineered high-affinity human prothrombinase complex.
Blood, 2025

PubMed Abstract: Thrombin is generated from prothrombin through cleavage at two sites by the enzyme prothrombinase, composed of factor (f) Xa and fVa. The affinity of fXa for fVa is low, with assembly and function dependent on phospholipid (PL) membranes. Some snakes have evolved venom versions of fXa that bind to fVa with high affinity and efficiently activate prothrombin in the absence of PL. We created a similar high-affinity, PL-independent human prothrombinase with 17 mutations to human fXa (M17). The increase in affinity enabled cryogenic electron microscopy (cryo-EM) structure determination of M17-prothrombinase to a resolution of 3.3 Å. All protein domains were well resolved in the map, except for the Gla domain of fXa. The main contacts involve the serine protease and EGF2 domains of fXa and the A2 and A3 domains of fVa, resulting in the burying of a total surface area of 4,900 Å2. The map is of sufficient quality to resolve side chain interactions, including several key M17 mutations. To aid in the placement of the loop C-terminal to the A2 domain (a2-loop), we solved a high-resolution crystal structure of fXa in complex with a synthetic a2-peptide. The acidic a2-loop interacts with the basic heparin binding site of fXa, involving a conserved antiparallel b-strand interaction. The M17-prothrombinase structure is compatible with data from biochemical and mutagenesis research, and provides important new insights into the assembly and function of the prothrombinase complex.

PubMed: 41144779
DOI: 10.1182/blood.2025031527

Experimental method

ELECTRON MICROSCOPY (3.28 Å)