SAXS analysis of the intrinsic tenase complex bound to a lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa

• Type of Material: Article
• Authors: Kenneth C Childers, Western Washington University; Shaun C Peters, Western Washington University; Pete Lollar, Emory University; H Trent Spencer, Emory University; Christopher Doering, Emory University; Paul C Spiegel, Western Washington University
• Language: English
• Date: 2022-06-14
• Publisher: ELSEVIER
• Publication Version: Final Published Version
• Copyright Statement: © 2022 by The American Society of Hematology.
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1182/bloodadvances.2021005874
• Title of Journal or Parent Work: BLOOD ADVANCES
• Volume: 6
• Issue: 11
• Start Page: 3240
• End Page: 3254
• Grant/Funding Information: Additional support comes from the National Institutes of Health project ALS-ENABLE (P30 GM124169) and a High-End Instrumentation Grant S10OD018483.; supported by DOE Office of Biological and Environmental Research.; This work was supported by the Dreyfus Foundation (Henry Dreyfus Teacher-Scholar Award) and the National Institutes of Health/National Heart, Lung and Blood Institute (award numbers R15HL103518 and U54HL141981 to P.C.S., award numbers R44HL117511, R44HL110448, U54HL112309, and U54HL141981 to C.B.D., H.T.S., and P.L.).
• Keywords: Hematology; FACTOR-X ACTIVATION; HIGH-LEVEL EXPRESSION; A2 SUBUNIT; Science & Technology; HEMOPHILIA-A; MEMBRANE-BINDING; COAGULATION-FACTOR-IX; BLOOD-COAGULATION; ACTIVATED FACTOR-VIII; GROWTH-FACTOR DOMAIN; Life Sciences & Biomedicine; PHOSPHOLIPID-BILAYER NANODISCS
• Research Categories: Chemistry, General

Abstract

The intrinsic tenase (Xase) complex, formed by factors (f) VIIIa and fIXa, forms on activated platelet surfaces and catalyzes the activation of factor X to Xa, stimulating thrombin production in the blood coagulation cascade. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering our understanding of the structural underpinnings that guide Xase complex assembly. Here, we aimed to characterize the Xase complex bound to a lipid nanodisc with biolayer interferometry (BLI), Michaelis-Menten kinetics, and small-angle X-ray scattering (SAXS). Using immobilized lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. Enzyme kinetic measurements demonstrated the assembly of an active enzyme complex in the presence of lipid nanodiscs. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein-protein interactions. Our results highlight multiple points of contact between fVIIIa and fIXa, including a novel interaction with fIXa at the fVIIIa A1-A3 domain interface. Lastly, we identified hemophilia A/B-related mutations with varying severities at the fVIIIa/fIXa interface that may regulate Xase complex assembly. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex.