TY - JOUR
T1 - An Asymmetric Runaway Domain Swap Antithrombin Dimer as a Key Intermediate for Polymerization Revealed by Hydrogen/Deuterium-Exchange Mass Spectrometry
AU - Trelle, Morten Beck
AU - Pedersen, Shona H
AU - Østerlund, Eva Christina
AU - Madsen, Jeppe Buur
AU - Kristensen, Søren Risom
AU - Jørgensen, Thomas J D
PY - 2017
Y1 - 2017
N2 - 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 domain swap mechanism for antithrombin polymerization. HDX-MS thus holds exceptional promise as an enabling analytical technique in the efforts towards future pharmacological intervention with protein polymerization and associated diseases.
AB - 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 domain swap mechanism for antithrombin polymerization. HDX-MS thus holds exceptional promise as an enabling analytical technique in the efforts towards future pharmacological intervention with protein polymerization and associated diseases.
U2 - 10.1021/acs.analchem.6b02518
DO - 10.1021/acs.analchem.6b02518
M3 - Journal article
C2 - 27783482
SN - 0003-2700
VL - 89
SP - 616
EP - 624
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 1
ER -