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Many regulatory processes in biology involve reversible association of proteins with membranes. Clotting proteins bind to phosphatidylserine (PS) on cell surfaces, but a clear picture of this interaction has yet to emerge. We present a novel explanation for membrane binding by GLA domains of clotting proteins, supported by biochemical studies, solid-state NMR analyses, and molecular dynamics simulations. The model invokes a single "phospho-L-serine-specific" interaction and multiple "phosphate-specific" interactions. In the latter, the phosphates in phospholipids interact with tightly bound Ca2+ in GLA domains. We show that phospholipids with any headgroup other than choline strongly synergize with PS to enhance factor X activation. We propose that phosphatidylcholine and sphin gomyelin (the major external phospholipids of healthy cells) are anticoagulant primarily because their bulky choline headgroups sterically hinder access to their phosphates. Following cell damage or activation, exposed PS and phosphatidylethanolamine collaborate to bind GLA domains by providing phospho-L-ser- me-specific and phosphate-specific interactions, respectively. [ABSTRACT FROM AUTHOR]

Membranes play key regulatory roles in biological processes, with bilayer composition exerting marked effects on binding affinities and catalytic activities of a number of membrane-associated proteins. In particular, proteins involved in diverse processes such as vesicle fusion, intracellular signaling cascades, and blood coagulation interact specifically with anionic lipids such as phosphatidylserine (PS) in the presence of Ca2+ ions. While Ca2+ is suspected to induce PS clustering in mixed phospholipid bilayers, the detailed structural effects of this ion on anionic lipids are not established. In this study, combining magic angle spinning (MAS) solid-state NMR (SSNMR) measurements of isotopically labeled serine headgroups in mixed lipid bilayers with molecular dynamics (MD) simulations of PS lipid bilayers in the presence of different counterions, we provide site-resolved insights into the effects of Ca2+ on the structure and dynamics of lipid bilayers. Ca2+-induced conformational changes of PS in mixed bilayers are observed in both liposomes and Nanodiscs, a nanoscale membrane mimetic of bilayer patches. Site-resolved multidimensional correlation SSNMR spectra of bilayers containing 13C,15N-labeled PS demonstrate that Ca2+ ions promote two major PS headgroup conformations, which are well resolved in two-dimensional 13C-13C, 15N-13C, and 31P-13C spectra. The results of MD simulations performed on PS lipid bilayers in the presence or absence of Ca2+ provide an atomic view of the conformational effects underlying the observed spectra. [ABSTRACT FROM AUTHOR]

Abstract: Most steps of the blood clotting cascade require the assembly of a serine protease with its specific regulatory protein on a suitable phospholipid bilayer. Unfortunately, the molecular details of how blood clotting proteins bind to membrane surfaces remain poorly understood, owing to a dearth of techniques for studying protein-membrane interactions at high resolution. Our laboratories are tackling this question using a combination of approaches, including nanoscale membrane bilayers, solid-state NMR, and large-scale molecular dynamics simulations. These studies are now providing structural insights at atomic resolution into clotting protein-membrane interactions. [Copyright &y& Elsevier]

Abstract: Blood clotting reactions, such as those catalyzed by the tissue factor:factor VIIa complex (TF:FVIIa), assemble on membrane surfaces containing anionic phospholipids such as phosphatidylserine (PS). In fact, membrane binding is critical for the function of most of the steps in the blood clotting cascade. In spite of this, our understanding of how the membrane contributes to catalysis, or even how these proteins interact with phospholipids, is incomplete. Making matters more complicated, membranes containing mixtures of PS and neutral phospholipids are known to spontaneously form PS-rich membrane microdomains in the presence of plasma concentrations of calcium ions, and it is likely that blood-clotting proteases such as TF:FVIIa partition into these PS-rich microdomains. Unfortunately, little is known about how membrane microdomain composition influences the activity of blood-clotting proteases, which is typically not under experimental control even in “simple” model membranes. Our laboratories have developed and applied new technologies for studying membrane proteins to gain insights into how blood-clotting protease–cofactor pairs assemble and function on membrane surfaces. This includes using a novel, nanoscale bilayer system (Nanodiscs) that permits assembling blood-clotting protease–cofactor pairs on stable bilayers containing from 65 to 250 phospholipid molecules per leaflet. We have used this system to investigate how local (nanometer-scale) changes in phospholipid bilayer composition modulate TF:FVIIa activity. We have also used detailed molecular-dynamics simulations of nanoscale bilayers to provide atomic-scale predictions of how the membrane-binding domain of factor VIIa interacts with PS in membranes. [Copyright &y& Elsevier]

Polyphosphates, linear polymers of inorganic phosphates linked by phosphoanhydride bonds, are widely present among organisms and play diverse roles in biology, including functioning as potent natural modulators of the human blood clotting system. However, studies of protein-polyphosphate interactions are hampered by a dearth of methods for derivatizing polyphosphate or immobilizing it onto solid supports. We now report that EDAC (1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide) efficiently promotes the covalent attachment of a variety of primary amine-containing labels and probes to the terminal phosphates of polyphosphates via stable phosphoramidate linkages. Using 31P NMR. we confirmed that EDAC-mediated reactions between primary amines and polyphosphate result in phosphoramidate linkages with the terminal phosphate groups. We show that polyphosphate can be biotinylated, labeled with fluorophores, and immobilized onto solid supports, that immobilized polyphosphate can be readily used to quantify protein binding affinities, that covalently derivatized or immobilized polyphosphate retains its ability to trigger blood clotting, and that derivatizing the ends of polyphosphate with spermidine protects it from exopolyphosphatase degradation. Our findings open up essentially the entire armamentarium of protein chemistry to modifying polyphosphate, which should greatly facilitate studies of its biological roles. [ABSTRACT FROM AUTHOR]