Divyani Paul1, James H Morrissey1,2. 1. Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan, USA. 2. Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Abstract
BACKGROUND: Cellular trauma or activation exposes phosphatidylserine (PS) and the substantially more abundant phospholipid, phosphatidylethanolamine (PE), on the outer layer of the plasma membrane, thereby allowing binding of many blood clotting proteins. We previously proposed the Anything But Choline (ABC) hypothesis to explain how PS and PE synergize to support binding of clotting proteins with gamma-carboxyglutamate (Gla)-rich domains, which posited that each Gla domain binds to a limited number of PS molecules and multiple PE molecules. However, the minimal number of PS molecules required to stably bind a Gla-domain-containing blood clotting protein in the presence of excess PE was unknown. OBJECTIVE: To test the ABC hypothesis for factor X by determining the threshold binding requirement of PS molecules under conditions of PS-PE synergy. METHODS: We used surface plasmon resonance to investigate the stoichiometry of factor X binding to nanoscale membrane bilayers (Nanodiscs) of varying phospholipid composition. RESULTS AND CONCLUSIONS: We quantified 1.05 ± 0.2 PS molecules per bound factor X molecule in Nanodiscs containing a mixture of 10% PS, 60% PE, and 30% phosphatidylcholine. Hence, there appears to be one truly PS-specific binding site per Gla domain, while the remaining membrane binding interactions can be satisfied by PE.
BACKGROUND: Cellular trauma or activation exposes phosphatidylserine (PS) and the substantially more abundant phospholipid, phosphatidylethanolamine (PE), on the outer layer of the plasma membrane, thereby allowing binding of many blood clotting proteins. We previously proposed the Anything But Choline (ABC) hypothesis to explain how PS and PE synergize to support binding of clotting proteins with gamma-carboxyglutamate (Gla)-rich domains, which posited that each Gla domain binds to a limited number of PS molecules and multiple PE molecules. However, the minimal number of PS molecules required to stably bind a Gla-domain-containing blood clotting protein in the presence of excess PE was unknown. OBJECTIVE: To test the ABC hypothesis for factor X by determining the threshold binding requirement of PS molecules under conditions of PS-PE synergy. METHODS: We used surface plasmon resonance to investigate the stoichiometry of factor X binding to nanoscale membrane bilayers (Nanodiscs) of varying phospholipid composition. RESULTS AND CONCLUSIONS: We quantified 1.05 ± 0.2 PS molecules per bound factor X molecule in Nanodiscs containing a mixture of 10% PS, 60% PE, and 30% phosphatidylcholine. Hence, there appears to be one truly PS-specific binding site per Gla domain, while the remaining membrane binding interactions can be satisfied by PE.
Authors: T K Ritchie; Y V Grinkova; T H Bayburt; I G Denisov; J K Zolnerciks; W M Atkins; S G Sligar Journal: Methods Enzymol Date: 2009 Impact factor: 1.600