title: An Asymmetric Runaway Domain Swap Antithrombin Dimer as a Key Intermediate for Polymerization Revealed by Hydrogen/Deuterium-Exchange Mass Spectrometry.
abstract: Antithrombin deficiency is associated with increased risk of venous thrombosis. In certain families, this condition is caused by pathogenic polymerization of mutated antithrombin in the blood. To facilitate future development of pharmaceuticals against antithrombin polymerization, an improved understanding of the polymerogenic intermediates is crucial. However, X-ray crystallography of these intermediates is severely hampered by the difficulty in obtaining well-diffracting crystals of transient and heterogeneous noncovalent protein assemblies. Furthermore, their large size prohibits structural analysis by NMR spectroscopy. Here, we show how hydrogen/deuterium-exchange mass spectrometry (HDX-MS) provides detailed insight into the structural dynamics of each subunit in a polymerization-competent antithrombin dimer. Upon deuteration, this dimer surprisingly yields bimodal isotope distributions for the majority of peptides, demonstrating an asymmetric configuration of the two subunits. The data reveal that one subunit is very dynamic, potentially intrinsically disordered, whereas the other is considerably less dynamic. The local subunit-specific deuterium uptake of this polymerization-competent dimer strongly supports a β4A-β5A β-hairpin runaway domain swap mechanism for antithrombin polymerization. HDX-MS thus holds exceptional promise as an enabling analytical technique in the efforts toward future pharmacological intervention with protein polymerization and associated diseases.
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark , Campusvej 55, 5230 Odense M, Denmark.
authors: Morten Beck
authors: MB
authors: Trelle
authors: Department of Clinical Biochemistry, Aalborg University Hospital , Hobrovej 18, 9000 Aalborg, Denmark.|Department of Clinical Medicine, Aalborg University , Sdr. Skovvej 15, 9000 Aalborg, Denmark.
authors: Shona
authors: S
authors: Pedersen
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark , Campusvej 55, 5230 Odense M, Denmark.
authors: Eva Christina
authors: EC
authors: Østerlund
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark , Campusvej 55, 5230 Odense M, Denmark.
authors: Jeppe Buur
authors: JB
authors: Madsen
authors: Department of Clinical Biochemistry, Aalborg University Hospital , Hobrovej 18, 9000 Aalborg, Denmark.|Department of Clinical Medicine, Aalborg University , Sdr. Skovvej 15, 9000 Aalborg, Denmark.
authors: Søren Risom
authors: SR
authors: Kristensen
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark , Campusvej 55, 5230 Odense M, Denmark.
authors: Thomas J D
authors: TJ
authors: Jørgensen
mesh_terms: D000991:Antithrombins / Q000737:chemistry*; D041961:Deuterium Exchange Measurement; D006801:Humans; D008958:Models, Molecular; D000072757:Protein Conformation, beta-Strand; D000072417:Protein Domains; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary
chemical_list: D000991:Antithrombins