1. Factor VIII-driven changes in activated factor IX explored by hydrogen-deuterium exchange mass spectrometry.
- Author
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Freato N, Ebberink EHTM, van Galen J, Fribourg C, Boon-Spijker M, van Alphen FPJ, Meijer AB, van den Biggelaar M, and Mertens K
- Subjects
- Allosteric Regulation genetics, Factor IXa genetics, Factor IXa metabolism, Factor VIII genetics, Factor VIII metabolism, Hemophilia B genetics, Hemophilia B metabolism, Humans, Protein Conformation, alpha-Helical, Protein Domains, Deuterium Exchange Measurement, Factor IXa chemistry, Factor VIII chemistry, Mass Spectrometry, Mutation
- Abstract
The assembly of the enzyme-activated factor IX (FIXa) with its cofactor, activated factor VIII (FVIIIa) is a crucial event in the coagulation cascade. The absence or dysfunction of either enzyme or cofactor severely compromises hemostasis and causes hemophilia. FIXa is a notoriously inefficient enzyme that needs FVIIIa to drive its hemostatic potential, by a mechanism that has remained largely elusive to date. In this study, we employed hydrogen-deuterium exchange-mass spectrometry (HDX-MS) to investigate how FIXa responds to assembly with FVIIIa in the presence of phospholipids. This revealed a complex pattern of changes that partially overlaps with those changes that occur upon occupation of the substrate-binding site by an active site-directed inhibitor. Among the changes driven by both cofactor and substrate, HDX-MS highlighted several surface loops that have been implicated in allosteric networks in related coagulation enzymes. Inspection of FVIIIa-specific changes indicated that 3 helices are involved in FIXa-FVIIIa assembly. These are part of a basic interface that is also known as exosite II. Mutagenesis of basic residues herein, followed by functional studies, identified this interface as an extended FVIIIa-interactive patch. HDX-MS was also applied to recombinant FIXa variants that are associated with severe hemophilia B. This revealed that single amino acid substitutions can silence the extended network of FVIIIa-driven allosteric changes. We conclude that HDX-MS has the potential to visualize the functional impact of disease-associated mutations on enzyme-cofactor complexes in the hemostatic system., (© 2020 by The American Society of Hematology.)
- Published
- 2020
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