An Electrostatic/Hydrogen Bond Switch as the Basis for the Specific Interaction of Phosphatidic Acid with Proteins

Phosphatidic acid (PA) is a minor but important phospholipid that, through specific interactions with proteins, plays a central role in several key cellular processes. The simple yet unique structure of PA, carrying just a phosphomonoester head group, suggests an important role for interactions with the positively charged essential residues in these proteins. We analyzed by solid-state magic angle spinning 31P NMR and molecular dynamics simulations the interaction of low concentrations of PA in model membranes with positively charged side chains of membrane-interacting peptides. Surprisingly, lysine and arginine residues increase the charge of PA, predominantly by forming hydrogen bonds with the phosphate of PA, thereby stabilizing the protein-lipid interaction. Our results demonstrate that this electrostatic/hydrogen bond switch turns the phosphate of PA into an effective and preferred docking site for lysine and arginine residues. In combination with the special packing properties of PA, PA may well be nature's preferred membrane lipid for interfacial insertion of positively charged membrane protein domains. Phosphatidic acid (PA) is a minor but important phospholipid that, through specific interactions with proteins, plays a central role in several key cellular processes. The simple yet unique structure of PA, carrying just a phosphomonoester head group, suggests an important role for interactions with the positively charged essential residues in these proteins. We analyzed by solid-state magic angle spinning 31P NMR and molecular dynamics simulations the interaction of low concentrations of PA in model membranes with positively charged side chains of membrane-interacting peptides. Surprisingly, lysine and arginine residues increase the charge of PA, predominantly by forming hydrogen bonds with the phosphate of PA, thereby stabilizing the protein-lipid interaction. Our results demonstrate that this electrostatic/hydrogen bond switch turns the phosphate of PA into an effective and preferred docking site for lysine and arginine residues. In combination with the special packing properties of PA, PA may well be nature's preferred membrane lipid for interfacial insertion of positively charged membrane protein domains. Phosphatidic acid (PA) 7The abbreviations used are: PA, phosphatidic acid; PE, phosphatidylethanol-amine; PC, phosphatidylcholine; DOPA, 1,2-dioleoyl-sn-glycero-3-phosphate (monosodium salt); DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; GST, glutathione S-transferase; LPA, lysophosphatidic acid; MAS, magic-angle spinning; RPA, Raf-1 PA binding region; MD, molecular dynamics. is a minor but important bioactive lipid involved in at least three essential and likely interrelated processes in a typical eukaryotic cell. PA is a key intermediate in the biosynthetic route of the main membrane phospholipids and triglycerides (1Athenstaedt K. Daum G. Eur. J. Biochem. 1999; 266: 1-16Crossref PubMed Scopus (271) Google Scholar); it is involved in membrane dynamics, i.e. fission and fusion (2Ktistakis N.T. Brown H.A. Waters M.G. Sternweis P.C. Roth M.G. J. Cell Biol. 1996; 134: 295-306Crossref PubMed Scopus (329) Google Scholar, 3Chen Y.G. Siddhanta A. Austin C.D. Hammond S.M. Sung T.C. Frohman M.A. Morris A.J. Shields D. J. Cell Biol. 1997; 138: 495-504Crossref PubMed Scopus (242) Google Scholar, 4Kooijman E.E. Chupin V. de Kruijff B. Burger K.N.J. Traffic. 2003; 4: 162-174Crossref PubMed Scopus (303) Google Scholar), and has important signaling functions (5English D. Cui Y. Siddiqui R.A. Chem. Phys. Lipids. 1996; 80: 117-132Crossref PubMed Scopus (158) Google Scholar, 6Freyberg Z. Siddhanta A. Shields D. Trends Cell Biol. 2003; 13: 540-546Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 7Testerink C. Munnik T. Trends Plant Sci. 2005; 10: 368-375Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar). The role of PA in membrane dynamics and signaling is most likely 2-fold, either via an effect on the packing properties of the membrane lipids or via the specific binding of effector proteins (4Kooijman E.E. Chupin V. de Kruijff B. Burger K.N.J. Traffic. 2003; 4: 162-174Crossref PubMed Scopus (303) Google Scholar, 7Testerink C. Munnik T. Trends Plant Sci. 2005; 10: 368-375Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 8Kooijman E.E. Carter K.M. van Laar E.G. Chupin V. Burger K.N. de Kruijff B. Biochemistry. 2005; 44: 17007-17015Crossref PubMed Scopus (137) Google Scholar, 9Kooijman E.E. Chupin V. Fuller N.L. Kozlov M.M. de Kruijff B. Burger K.N.J. Rand P.R. Biochemistry. 2005; 44: 2097-2102Crossref PubMed Scopus (219) Google Scholar). The origin of the specific binding of effector proteins to PA is not known. The PA binding domains thus far identified (for review, see Ref. 10Andresen B.T. Rizzo M.A. Shome K. Romero G. FEBS Lett. 2002; 531: 65-68Crossref PubMed Scopus (157) Google Scholar) and more recently (7Testerink C. Munnik T. Trends Plant Sci. 2005; 10: 368-375Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar)) are diverse and share no apparent sequence homology, in contrast to other lipid binding domains, such as e.g. the PH, PX, FYVE, and C2 domains (11Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar, 12Maffucci T. Falasca M. FEBS Lett. 2001; 506: 173-179Crossref PubMed Scopus (110) Google Scholar, 13Ellson C.D. Andrews S. Stephens L.R. Hawkins P.T. J. Cell Sci. 2002; 115: 1099-1105Crossref PubMed Google Scholar, 14Stenmark H. Aasland R. Driscoll P.C. FEBS Lett. 2002; 513: 77-84Crossref PubMed Scopus (158) Google Scholar, 15Lemmon M.A. Traffic. 2003; 4: 201-213Crossref PubMed Scopus (488) Google Scholar). One general feature that the PA binding domains do have in common is the presence of basic amino acids (7Testerink C. Munnik T. Trends Plant Sci. 2005; 10: 368-375Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar). Where examined in detail, these basic amino acids were shown to be essential for the interaction with PA, which underscores the importance of electrostatic interactions. Indeed, the negatively charged phosphomonoester head group of PA would be expected to interact electrostatically with basic amino acids in a lipid binding domain. However, such a simple electrostatic interaction cannot explain the strong preference of PA-binding proteins for PA over other, often more abundant, negatively charged phospholipids. In a recent study we showed that the phosphomonoester head group of PA has remarkable properties (8Kooijman E.E. Carter K.M. van Laar E.G. Chupin V. Burger K.N. de Kruijff B. Biochemistry. 2005; 44: 17007-17015Crossref PubMed Scopus (137) Google Scholar). The phosphomonoester head group is able to form an intramolecular hydrogen bond upon initial deprotonation (when its charge is -1), which stabilizes the second proton against dissociation. We provided evidence that competing hydrogen bonds, e.g. from the primary amine of the head group of phosphati-dylethanolamine (PE), can destabilize this intramolecular hydrogen bond and, thus, favor the further deprotonation, i.e. increase the negative charge, of PA (8Kooijman E.E. Carter K.M. van Laar E.G. Chupin V. Burger K.N. de Kruijff B. Biochemistry. 2005; 44: 17007-17015Crossref PubMed Scopus (137) Google Scholar). These data raised the intriguing hypothesis that a combination of electrostatic and hydrogen bond interactions and not just electrostatic interactions of PA with basic amino acids, i.e. lysine and arginine, forms the basis of the (specific) binding of PA to PA-binding proteins. We set out to this hypothesis by the of lysine and arginine residues in membrane-interacting to form a hydrogen bond with the phosphomonoester head group of PA in a lipid with we magic angle spinning 31P NMR that the positively charged amino acids lysine and arginine in these are able to increase the charge of We that this increase in charge is to the of hydrogen bonds PA and the basic amino simulations provided a on the specific docking of these side chains on the form of We that the phosphate of PA is an effective and preferred docking site for lysine and arginine residues in membrane-interacting peptides. In combination with its special packing this turns PA into nature's preferred membrane lipid to interfacial insertion of positively charged membrane protein domains. preference for PA and the role of interfacial insertion in binding with the PA binding of the protein which is of the PA proteins S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, M.A. Shome K. C. Sung T.C. Frohman M.A. Romero G. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M.A. Shome K. Romero G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). we that the electrostatic/hydrogen bond switch provided by the phosphomonoester is a in stabilizing the binding of to proteins. (monosodium and were from lipids a from M. of The The and were from by and to be more were as and were more as by Biochemistry. 2002; PubMed Scopus Google Scholar); with of and were from and used further lipid and lipid were as (8Kooijman E.E. Carter K.M. van Laar E.G. Chupin V. Burger K.N. de Kruijff B. Biochemistry. 2005; 44: 17007-17015Crossref PubMed Scopus (137) Google Scholar). were by the lipid with and The were at least which the of the to be of the to this The were at for at in a and the to for 31P NMR were as Biochemistry. 2002; PubMed Scopus Google Scholar). These were with and the were as The and the in and the of the of which the were at least the of this of it and the the the in the NMR NMR were on a at a were at the magic angle at to the and the of PA to spinning were were out at a of the with of and 2005; of structure were that the lysine i.e. primary hydrogen bond the arginine i.e. hydrogen bond structure were were with the P.R. M. J. R. B. 2002; PubMed Scopus Google Scholar); for data the for the and of from the Scholar) for molecular dynamics simulations were on simulations of interactions and used the Biochemistry. 2003; PubMed Scopus Google Scholar). The of in membrane In were into or by the head group and for and lipids were and The were to the phosphate group in and in to the The and and The DOPC, and These were for for as by in the lipid and hydrogen and for and were in an with were to the of the by and to a charge in the the were such that no with lipids at but in the at the and showed in the by the of the of of the in the and hydrogen the and were as over the of the hydrogen bonds were by the of a of and an angle of simulations and were with B. van D. J. 2001; Google Scholar); with A. K. J. 1996; PubMed Scopus Google Scholar). fusion of the Raf-1 PA binding acids S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) in The by and into the the The to and by to the protein and protein were as S. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, C. N.T. PubMed Scopus Google Scholar) with in of phospholipids from were at the in and in for were a lipid to the were in of binding and by at for were in binding to of and for in a of at were by at for in binding and in were on and were with and with with in the of 31P of PA to be to the of the phosphate head in to e.g. by an increase in to an increase in negative charge either via NMR in membrane M. de Kruijff B. van PubMed Scopus Google Scholar, H. Sci. S. A. PubMed Scopus Google Scholar) or by NMR in more (8Kooijman E.E. Carter K.M. van Laar E.G. Chupin V. Burger K.N. de Kruijff B. Biochemistry. 2005; 44: 17007-17015Crossref PubMed Scopus (137) Google Scholar). In an study we that of in a in a of the 31P NMR of PA (8Kooijman E.E. Carter K.M. van Laar E.G. Chupin V. Burger K.N. de Kruijff B. Biochemistry. 2005; 44: 17007-17015Crossref PubMed Scopus (137) Google Scholar). is in and the 31P NMR of a with the minor the to PA and the the to of results in a of the PA to to an increase in the charge of increase in negative charge of PA by is by hydrogen the primary amine in the head group of and the phosphomonoester head group of PA 8Kooijman E.E. Carter K.M. van Laar E.G. Chupin V. Burger K.N. de Kruijff B. Biochemistry. 2005; 44: 17007-17015Crossref PubMed Scopus (137) Google Scholar). the basic amino acid which a primary is able to form a hydrogen bond with PA, we the effect of on the of PA at with a of it with to membranes negatively charged phospholipid Kruijff B. A. B. PubMed Scopus Google Scholar, J. M. H. S. J. Full Text PDF PubMed Scopus Google Scholar). that an of lysine residues with to PA the of the PA to to The minor of the PA that not upon most likely a of PA in the that is not by In the presence of an of lysine residues with to PA the the PA further for These results were not at the of results in an increase in the negative charge of The binding of to negatively charged lipid membranes is and a of at least lysine residues Kruijff B. A. B. PubMed Scopus Google Scholar). In the PA binding domains that have identified to a of basic amino acids in PA binding site (7Testerink C. Munnik T. Trends Plant Sci. 2005; 10: 368-375Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 10Andresen B.T. Rizzo M.A. Shome K. Romero G. FEBS Lett. 2002; 531: 65-68Crossref PubMed Scopus (157) Google Scholar). more the we a well of on either side by basic amino acids to study the interaction of of the basic amino acids, and with PA Biochemistry. 2002; PubMed Scopus Google Scholar, de Kruijff B. Biochemistry. 2001; PubMed Scopus Google Scholar). that at a of a of the PA in that is able to increase the charge of PA, to the of and The effect of on the charge of PA on the as is shown in results were with a not which has a the de Kruijff B. Biochemistry. 2001; PubMed Scopus Google Scholar). an increase in the negative charge of PA in the a we the effect of the at concentrations not the of PA and and the a not the positively charged lysine and arginine residues in and are for the charge increase of PA and not just the presence of a The in to from the of forming a hydrogen bond with the phosphomonoester head group of PA, lysine and arginine a charge at the membrane the proton to charge and, thus, increase the increase in the interfacial in an increase in the negative charge of PA and is by a of the PA charge and hydrogen bond we a in which to this We the positively charged and these the charge but in the to form hydrogen bonds the amine cannot as a hydrogen bond and were in the at a of and to be able to the charge by these with by the and the results are shown in the positively charged the charge of PA to the interaction of a charge results were for the amine and which a increase in the and, thus, the negative charge of PA not However, which in to positively charged can form a hydrogen a increase in the negative charge of PA of the in charge by primary and which are shown in These data that at primary an increase in the negative charge with These results demonstrate that hydrogen primary are able to further increase the negative charge of PA the increase by and that the increase in negative charge by the primary amine is to the of a hydrogen bond with the phosphomonoester head group of the by the are by and the effect of the primary amine is to that of and we that the interaction of and with PA the of hydrogen bonds the basic amino acid residues and the phosphomonoester head group of a with a into the interaction of lysine and arginine membrane with a we out These simulations were with of either a or PA and either or on either side of the lipid The of these is to an interaction with an Kruijff B. A. B. PubMed Scopus Google Scholar). The simulations were with the in and we the interaction lysine or arginine side chains and in the head group of PA and The to the lipid and for the of the of the of a in the presence of is shown in by the hydrogen bond interactions the PA and lysine side chains are in However, hydrogen bonds the phosphate of and lysine side chains were an of the preference of the lysine side chains for a specific lipid PA or we the of hydrogen bonds the and phosphate of and PA and and these to the of PA to in the The of lysine hydrogen bonds to the phosphate of PA and in the simulations either or PA to the in the are shown in and These that it is more likely for a lysine side to form a hydrogen bond with the phosphate of PA with the phosphate of in is no apparent PA and PC, i.e. the These that over is the not from the of the hydrogen bond interactions of a of the a of the is in the The hydrogen bond interaction lysine side chains and PA is with hydrogen bond well over not simulations were with the and an hydrogen bond The for the and PA simulations are shown in and These that arginine side chains are more likely to form a hydrogen bond with the phosphate of PA PC, an of the and that is no preference or PA and of the of hydrogen bonds lysine side chains and the phosphate of and PA in the and simulations and arginine side chains and the phosphate of and PA in the and to the in the lipid bonds to are in and the of the and to PA in and the of the of a PA by unique of the phosphate head group of PA, to the of the lipid turns PA into an for interactions. PA is a i.e. has a (4Kooijman E.E. Chupin V. de Kruijff B. Burger K.N.J. Traffic. 2003; 4: 162-174Crossref PubMed Scopus (303) Google Scholar, 8Kooijman E.E. Carter K.M. van Laar E.G. Chupin V. Burger K.N. de Kruijff B. Biochemistry. 2005; 44: 17007-17015Crossref PubMed Scopus (137) Google Scholar), and this of PA favor interactions PA and PA binding domains de Kruijff B. PubMed Scopus Google Scholar). Indeed, most PA binding domains essential to for such interactions. We the effect of negative on the binding of the well PA binding of the protein Raf-1 S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, M.A. Shome K. C. Sung T.C. Frohman M.A. Romero G. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M.A. Shome K. Romero G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). this in model membranes by PE, and protein binding The results that of binding of the Raf-1 PA domain. We not in with that binding is specific for PA but that PA is in the presence of concentrations of results were for the PA-binding proteins and The of PA an for that the positively charged amino acids lysine and arginine in the membrane-interacting and a in the 31P NMR of PA at We showed that the main this increase in negative charge of PA is hydrogen bond the lysine or arginine side chains and the phosphomonoester head group of The of hydrogen bonds the and phosphate of PA and lysine and arginine side chains further by a effect on PA charge is of the lysine residues are from the as or are in the membrane as is the in the and The of the and the of the de Kruijff B. Biochemistry. 2001; PubMed Scopus Google Scholar) and thereby the lysine residues in the group the phosphate group of PA is The of the phosphate of PA by of a and The results that the and phosphate of PA is at the as the of in the head group the lysine residues in and an of the PA an effect on the negative charge of is the that is with a that is the of a de Kruijff B. Biochemistry. 2001; PubMed Scopus Google Scholar). on in that the lysine residues in are in the head group of the lipid and the of the to that of the see Ref. Biochemistry. 2002; PubMed Scopus Google Scholar). in the presence of PA, the to the PA phosphate of the head group that the phosphomonoester head group of PA as an effective docking site for the lysine residues. the of hydrogen bonds lysine residues and PA, as shown by the and with docking of the membrane-interacting on the phosphomonoester head group of PA and not an electrostatic into the over the lipid head showed that the most effective docking on a PA, with 31P NMR that the interaction of lysine residues with the phosphate of PA its charge from at The interaction PA and lysine side chains is by a proton from to a PA from the phosphomonoester head group of PA, which in the presence of a positively charged side increase in PA charge by arginine residues in is that by the lysine residues in The of this is but may be to the of charge in the group of arginine with the primary amine of its hydrogen bond is by a of the data for the hydrogen bond of lysine and arginine to a phosphate which that the hydrogen bond for the lysine is that of the see The simulations of and with a further these results and showed that the hydrogen bond arginine and the PA phosphate is that for lysine to PA These results that the group of arginine is a hydrogen bond the primary amine of the data to the for the interaction of lysine and arginine residues in membrane-interacting with low concentrations of PA in a The charge of lysine and arginine side chains in these results in an electrostatic to the negatively charged phospholipid binding to the the positively charged side chains are able to i.e. interact electrostatically and form hydrogen bonds with negatively charged However, as as the side of lysine or arginine the phosphomonoester head group of PA and into to form a hydrogen the negative charge of PA to the further deprotonation of its phosphomonoester head We this the electrostatic/hydrogen bond The increase in negative charge, the electrostatic to the of hydrogen bonds the positively charged lysine or arginine side chains on the head group of PA and results in a docking of the membrane-interacting on a PA The in hydrogen bond and effect on the negative charge of PA lysine and arginine residues would that lysine residues are more effective in docking on the phosphomonoester head group of We that the electrostatic/hydrogen bond switch is a key of the specific of PA by PA-binding proteins. for model from the interaction of basic residues in proteins with phospholipids and from the interaction of lysophosphatidic acid with its important feature of proteins is that are on the side of the membrane by basic amino These basic residues are to the of the protein its G. de Kruijff B. J. 1997; PubMed Scopus Google Scholar, G. Trends Biochem. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. Kozlov M.M. Cell Biol. 2005; Scopus Google Scholar). of these basic amino acids may to Indeed, recent evidence suggests that this is the for the of a of three basic amino acids and on its and has a interaction with PA Biochemistry. 2005; 44: PubMed Scopus Google Scholar). PA has a phosphomonoester head group, and binding of to its is likely to on as for Indeed, the binding of its to its on the phosphomonoester head group 2002; PubMed Scopus Google Scholar, K. T. G. 2002; PubMed Scopus Google Scholar), and the phosphate binding of the basic residues arginine and lysine K. T. G. 2002; PubMed Scopus Google results and the would that to its in a are in not in lipids such as PA and but in proteins, often and as well as in bioactive such as and binding to the phosphomonoester of these is likely by as for PA a of the structure of proteins binding a phosphomonoester or that hydrogen bonds are amino acid side chains and the which negative G. M.M. Full Text Full Text PDF PubMed Scopus Google Scholar, C. 1998; Full Text Full Text PDF PubMed Scopus (110) Google Scholar, M. R. M. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, K.M. M.A. Full Text Full Text PDF PubMed Scopus Google Scholar, S. T. J. A. Full Text Full Text PDF PubMed Scopus Google Scholar, J. D. C.D. Hawkins P.T. Stephens 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, D. S. D. S. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, T. M. 2001; PubMed Scopus Google Scholar, M. van Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, M.A. Z. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. H. J. C. J. Biol. PubMed Scopus Google Scholar, Sci. 2005; PubMed Scopus Google Scholar, R. J. C. M. Sci. S. A. 2005; PubMed Scopus Google Scholar). and arginine residues form the positively charged binding and often form the hydrogen bond as but other residues side hydrogen bonds We that the electrostatic/hydrogen bond switch is the by which are and by proteins. PA the for the of docking of basic protein domains on PA may be by insertion of protein domains into the of the lipid One of such a interaction has in for the which to negatively charged more insertion in PA of other negatively charged phospholipids molecular of insertion is in the presence of see Ref. K.N.J. R.A. de Kruijff B. Biochemistry. PubMed Scopus Google Scholar). can we these of its charge and to form hydrogen bonds, PA has a special molecular 4Kooijman E.E. Chupin V. de Kruijff B. Burger K.N.J. Traffic. 2003; 4: 162-174Crossref PubMed Scopus (303) Google and 9Kooijman E.E. Chupin V. Fuller N.L. Kozlov M.M. de Kruijff B. Burger K.N.J. Rand P.R. Biochemistry. 2005; 44: 2097-2102Crossref PubMed Scopus (219) Google see PA is the phospholipid with a J. Kozlov M.M. Cell Biol. 2005; Scopus Google Scholar). lipids protein into the membrane by forming insertion in the head group of the lipid de Kruijff B. PubMed Scopus Google Scholar). We further the effect of lipids on the binding of PA binding proteins to PA in with the well PA binding of the protein Raf-1 The lipid to increase PA binding of the PA binding of binding on the presence of insertion not to a binding of the and at least more to PA with in the presence of concentrations of Our that PA may as a docking site for membrane-interacting to the of the lipid with the of PA turns PA into a effective insertion site for positively charged proteins. We that the electrostatic/hydrogen bond switch in the phosphate head group of PA to the of the phosphate head group to the of the lipid PA from the other membrane in recent study of the has shown that PA and forms hydrogen bonds its phosphomonoester and arginine residues in the the the C. J. Full Text Full Text PDF PubMed Scopus (66) Google Scholar). with this PA to and the against R. B. de and J. A. We and for and on the and properties of the peptides. is for on the data with

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.