Conformation of factor Xa in solution revealed by single-molecule spectroscopy

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biophysics

Background:

• Existing X-ray structures of Factor Xa (FXa) lack the γ-carboxyglutamate (Gla) domain, preventing visualization of the complete protein conformation.
• The sole existing full-length FXa structure, from cryo-electron microscopy (cryo-EM) of the prothrombinase complex, indicates a specific Gla domain positioning.

Purpose of the Study:

• To determine if the curved conformation of FXa observed in cryo-EM is also present in solution.
• To characterize the solution conformation of full-length FXa.

Main Methods:

• Single-molecule Förster resonance energy transfer (smFRET) was employed to study FXa conformation in solution.
• Conformational analysis was performed on free FXa and FXa bound to physiological substrates.

Main Results:

• The solution conformation of full-length FXa was resolved for the first time, revealing a curved structure.
• FXa conformation in solution is highly sensitive to calcium ions (Ca2+).
• The solution conformation is similar to its zymogen form and its conformation within the prothrombinase complex, whether free or bound to prothrombin or meizothrombin.

Conclusions:

• Single-molecule Förster resonance energy transfer confirms FXa possesses a curved conformation in solution, both free and when bound to ligands, validating cryo-EM findings.
• Significant conformational changes in the absence of Ca2+ suggest that vitamin K antagonists, by affecting Gla domain interactions with divalent cations, alter FXa's structural architecture.