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Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker

Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor (VWF) to GPIbα on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from fo...

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Autores principales: Bonazza, Klaus, Iacob, Roxana E, Hudson, Nathan E, Li, Jing, Lu, Chafen, Engen, John R, Springer, Timothy A
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9084892/
https://www.ncbi.nlm.nih.gov/pubmed/35532124
http://dx.doi.org/10.7554/eLife.75760
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author Bonazza, Klaus
Iacob, Roxana E
Hudson, Nathan E
Li, Jing
Lu, Chafen
Engen, John R
Springer, Timothy A
author_facet Bonazza, Klaus
Iacob, Roxana E
Hudson, Nathan E
Li, Jing
Lu, Chafen
Engen, John R
Springer, Timothy A
author_sort Bonazza, Klaus
collection PubMed
description Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor (VWF) to GPIbα on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from force-induced unfolding by a long-range disulfide that links cysteines near its N- and C-termini. The O-glycosylated linkers between A1 and its neighboring domains, which transmit tensile force to A1, are reported to regulate A1 activation for binding to GPIb, but the mechanism is controversial and incompletely defined. Here, we study how these linkers, and their polypeptide and O-glycan moieties, regulate A1 affinity by measuring affinity, kinetics, thermodynamics, hydrogen deuterium exchange (HDX), and unfolding by temperature and urea. The N-linker lowers A1 affinity 40-fold with a stronger contribution from its O-glycan than polypeptide moiety. The N-linker also decreases HDX in specific regions of A1 and increases thermal stability and the energy gap between its native state and an intermediate state, which is observed in urea-induced unfolding. The C-linker also decreases affinity of A1 for GPIbα, but in contrast to the N-linker, has no significant effect on HDX or A1 stability. Among different models for A1 activation, our data are consistent with the model that the intermediate state has high affinity for GPIbα, which is induced by tensile force physiologically and regulated allosterically by the N-linker.
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spelling pubmed-90848922022-05-10 Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker Bonazza, Klaus Iacob, Roxana E Hudson, Nathan E Li, Jing Lu, Chafen Engen, John R Springer, Timothy A eLife Structural Biology and Molecular Biophysics Hemostasis in the arterial circulation is mediated by binding of the A1 domain of the ultralong protein von Willebrand factor (VWF) to GPIbα on platelets to form a platelet plug. A1 is activated by tensile force on VWF concatemers imparted by hydrodynamic drag force. The A1 core is protected from force-induced unfolding by a long-range disulfide that links cysteines near its N- and C-termini. The O-glycosylated linkers between A1 and its neighboring domains, which transmit tensile force to A1, are reported to regulate A1 activation for binding to GPIb, but the mechanism is controversial and incompletely defined. Here, we study how these linkers, and their polypeptide and O-glycan moieties, regulate A1 affinity by measuring affinity, kinetics, thermodynamics, hydrogen deuterium exchange (HDX), and unfolding by temperature and urea. The N-linker lowers A1 affinity 40-fold with a stronger contribution from its O-glycan than polypeptide moiety. The N-linker also decreases HDX in specific regions of A1 and increases thermal stability and the energy gap between its native state and an intermediate state, which is observed in urea-induced unfolding. The C-linker also decreases affinity of A1 for GPIbα, but in contrast to the N-linker, has no significant effect on HDX or A1 stability. Among different models for A1 activation, our data are consistent with the model that the intermediate state has high affinity for GPIbα, which is induced by tensile force physiologically and regulated allosterically by the N-linker. eLife Sciences Publications, Ltd 2022-05-09 /pmc/articles/PMC9084892/ /pubmed/35532124 http://dx.doi.org/10.7554/eLife.75760 Text en © 2022, Bonazza et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Structural Biology and Molecular Biophysics
Bonazza, Klaus
Iacob, Roxana E
Hudson, Nathan E
Li, Jing
Lu, Chafen
Engen, John R
Springer, Timothy A
Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker
title Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker
title_full Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker
title_fullStr Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker
title_full_unstemmed Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker
title_short Von Willebrand factor A1 domain stability and affinity for GPIbα are differentially regulated by its O-glycosylated N- and C-linker
title_sort von willebrand factor a1 domain stability and affinity for gpibα are differentially regulated by its o-glycosylated n- and c-linker
topic Structural Biology and Molecular Biophysics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9084892/
https://www.ncbi.nlm.nih.gov/pubmed/35532124
http://dx.doi.org/10.7554/eLife.75760
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