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Vitamin K–dependent proteins play a central role in blood coagu-lation and its regulation1,2. These proteins have N-terminal γ-carboxy-glutamic acid (Gla)-containing domains that anchor the proteins, in aCa2+-dependent interaction, to cell membranes exposing phosphat-idylserine3–5. The Gla domain–membrane interaction has a critical biological role in assembly of protein complexes with enhancedcatalytic efficiency6. Unlike many globular protein domains in whichhydrophobic packing within the core of the protein domain stabilizesthe structure, the tertiary fold of the Gla domain is stabilized by a linear array of internal Ca2+ ions bound by Gla side chain carboxylgroups7−12. Calcium ion binding to the Gla domain induces a markedstructural transition from a largely unfolded and nonfunctionaldomain to one that is tightly folded and able to bind mem-branes7,8,13,14. A solvent-exposed hydrophobic patch is present in theN terminus of Ca2+-liganded Gla domains (such as Phe5, Leu6 andVal9 in bovine prothrombin)7−12. These residues reside in a surfaceloop termed the ω-loop. Several studies have demonstrated a role forthe solvent-exposed hydrophobic residues within the ω-loop region inphospholipid membrane binding15−18. However, the interaction ofthese hydrophobic residues with the membrane surface does not pro-vide a basis for the phosphatidylserine specificity of Gladomain–membrane binding. To determine the basis of this specificitywe have solved the structure of bovine prothrombin fragment 1, PT1(Fig. 1a), in complex with Ca2+ ions and lysophosphatidylserine. Thisternary complex is compared with the binary complex containing PT1and Ca2+ ions. We have also explored the interactions of lysophos-phatidylserine with PT(1–46) in solution. PT(1–46) is a selectively

15N-labeled peptide based on the sequence of the human prothrombinGla domain (Fig. 1a).

RESULTSComplex of PT1, Ca2+ ions and lysophosphatidylserineThe overall tertiary structure of PT1 within the ternary complex of PT1,Ca2+ ions and lysophosphatidylserine is very similar to that of the binaryPT1–Ca2+ complex determined in this work (r.m.s. deviation 0.21 Å) (Table 1) and previously7 (Fig. 1b). Lysophosphatidylserine isbound along one face of the Gla domain with the serine head group ori-ented toward Gla21, the glycerol backbone adjacent to Phe5 and Leu6,and the SN1 acyl chain oriented toward the side chains of Phe5 and Leu6(Fig. 1b, inset). There is extensive interaction of lysophosphatidylserinewith the N-terminal region of the Gla domain with 40% of the surface oflysophosphatidylserine buried within the Gla domain of PT1 (1.4 Åprobe)19.

The head group of lysophosphatidylserine is anchored by interac-tion with protein-bound Ca2+ ions (Fig. 2a). The carboxyl oxygens ofthe serine head group are ligands for Ca5 and Ca6, displacing boundwater from the coordination spheres of each Ca2+ ion in the binarycomplex. The serine amino group is an indirect ligand for Ca6 througha hydrogen bond to a water molecule. In addition, a serine carboxyl oflysophosphatidylserine forms hydrogen bonds with a carboxyl oxygenof Gla17 and Gla21 (Fig. 1b, inset).

The glycerophosphate backbone binds to a positively charged patchformed by Lys3, Arg10 and Arg16 on one face of the Gla domain(Fig. 2b). There is a salt bridge and a hydrogen bond between the ter-

1Center for Hemostasis and Thrombosis Research, Beth Israel Deaconess Medical Center and Department of Medicine, Harvard Medical School, 330 BrooklineAvenue, Boston, Massachusetts 02215, USA. 2Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston,Massachusetts 02118, USA. 3These two authors contributed equally to this article. Correspondence should be addressed to B.C.F. (bfurie1@bidmc.harvard.edu).

Published online 17 August 2003; doi:10.1038/nsb971

Structural basis of membrane binding by Gla domains ofvitamin K–dependent proteinsMingdong Huang1,3, Alan C Rigby1,3, Xavier Morelli1, Marianne A Grant1, Guiqing Huang1, Bruce Furie1,Barbara Seaton2 & Barbara C Furie1

In a calcium-dependent interaction critical for blood coagulation, vitamin K–dependent blood coagulation proteins bind cellmembranes containing phosphatidylserine via �-carboxyglutamic acid–rich (Gla) domains. Gla domain–mediated protein-membrane interaction is required for generation of thrombin, the terminal enzyme in the coagulation cascade, on a physiologictime scale. We determined by X-ray crystallography and NMR spectroscopy the lysophosphatidylserine-binding site in the bovineprothrombin Gla domain. The serine head group binds Gla domain–bound calcium ions and Gla residues 17 and 21, fixedelements of the Gla domain fold, predicting the structural basis for phosphatidylserine specificity among Gla domains. Gladomains provide a unique mechanism for protein-phospholipid membrane interaction. Increasingly Gla domains are beingidentified in proteins unrelated to blood coagulation. Thus, this membrane-binding mechanism may be important in otherphysiologic processes.

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minal phosphate oxygens and the NH1 of Arg10 and a salt bridge and ahydrogen bond between a terminal phosphate oxygen and the Nε andNH1 of Arg16 (Fig. 2a). The Nζ of Lys3 forms two hydrogen bonds anda salt bridge with the acyl and phosphoryl ester oxygens of the glyc-erophosphate backbone (Fig. 2a). The glycerol backbone is in van derWaals contact with the protein backbone at Leu6 and Gla7 whereas theSN1 acyl chain is in van der Waals contact with Phe5 and the side chainof Leu6. There are hydrogen bonds between the amide nitrogen atomsof Leu6 and nitrogen atoms of Gla7 and the acyl and hydroxyl oxygensof the glycerophosphate backbone, respectively (Fig. 2a).

Phosphatidylserine binding to Arg10 and Arg16NMR experiments using selectively 15N labeled PT(1–46) confirm theimportance of Arg10 (bovine prothrombin numbering) in lysophos-

phatidylserine binding. In 2D 1H-15N heteronuclear single quantumcorrelation (HSQC) NMR experiments with PT(1–46) in complexwith Ca2+ ions we observe resonances for the amide NH and the NHεof Arg10 (Fig. 3a). The hydrogens bound to the Nη atoms of Arg10 areexchanging rapidly with the bulk solvent and resonances are notobserved. In the presence of Ca2+ ions the NHε of Arg10 in PT(1–46)gives rise to a single resonance peak at 6.89 p.p.m. With the addition ofincreasing concentrations of lysophosphatidylserine, the NHε reso-nance of Arg10 shifts from 6.89 p.p.m. to 7.06 p.p.m. and undergoesexchange broadening (Fig. 3b) indicating a perturbation in the chem-ical environment of the NHε upon lysophosphatidylserine binding. Incontrast, when 0.8 equivalents of lysophosphatidylcholine are addedto PT(1–46) there is no shift in the NHε resonance of Arg10. Thedownfield shift observed in the presence of lysophosphatidylserine is

752 VOLUME 10 NUMBER 9 SEPTEMBER 2003 NATURE STRUCTURAL BIOLOGY

Figure 1 Structure of the PT1–Ca2+–lysophosphatidylserine complex. (a) The aminoacid sequences of PT1, the Gla domain andfirst kringle domain of bovine prothrombin,and the human prothrombin Gla domain arealigned. (b) The Cα-trace of PT1 in the ternarycomplex derived from X-ray crystallographicdata is shown with the backbone of amino acidresidues 1–156 colored from blue to red. TheGla domain includes residues 1–47. Theconformations of the carbohydrate chainsbound at Asn77 and Asn101 in the kringledomain are indicated. Disulfide bonds are ingreen. The structure of lysophosphatidylserinewithin the complex is also shown: nitrogen,blue; oxygen, red; carbon, gray; phosphorous,green. Bound calcium atoms are blackspheres. Inset: expanded view, in stereo, of thestructure of the ternary complex includingbackbone for PT1 residues 1–33. Electrondensity for lysophosphatidylserine is indicated.Figures 1b, 2 and 3c were prepared usingMolScript42, BobScript43 and Raster3D44.

a

b

Figure 2 Bonding network for binding of lysophosphatidylserine with Ca2+-liganded PT1. (a) The model is rotated 90° around the y-axis relative to the view inFigure 1b. The hydrogen bonds and salt bridges between atoms in lysophosphatidylserine and the PT1–calcium ion complex are indicated as dashed lines.(b) The electrostatic potential surface of bovine PT1, generated using GRASP45, is shown in the same view as in a. Positive and negative electrostaticpotential on the surface, blue and red, respectively. Lysophosphatidylserine is presented as a stick model. Ca2+ ions are black spheres.

a b

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likely due to the formation of polar bonds between the NH1 and theterminal phosphate oxygens, although this is not observed on theNMR timescale (Fig. 2a). The hydrogen bond to the terminal phos-phate oxygen replaces a hydrogen bond from NH1 to a bound watermolecule in the binary complex of PT1 and Ca2+ ions.

In the same HSQC experiment, we observe a resonance for theamide NH, the NHε and a single crosspeak for the NHη groups ofArg16 (Fig. 3a). Rapid rotational mobility around the Nε–Cδ bondcontributes to conformational heterogeneity resulting in a singledegenerate NHη at 6.76 p.p.m20. On addition of lysophosphatidyl-serine to the complex of PT (1–46) and Ca2+ ions there is only a mod-est chemical shift in the degenerate resonance for the NHη groups. Inthe crystal structure of the ternary complex we observe a salt bridgeand a hydrogen bond between a terminal phosphate oxygen oflysophosphatidylserine and the NH1 and Nε of Arg16; however, thedistances of interaction between Arg16 and the glycerophosphatebackbone are greater than for Arg10 and the glycerophosphate back-bone. In addition, the Arg16 side chain is involved in a more extensivenetwork of stabilizing interactions in the binary complex than isArg10. In PT1 complexed with Ca2+ ions, the NH1 of Arg16 forms ahydrogen bond with the backbone carbonyl oxygen of Lys11. The sidechain of Arg16 is less solvent exposed in the binary complex than is theside chain of Arg10 and is impacted less by the addition of lysophos-phatidylserine.

Alterations in the �-loop upon phosphatidylserine bindingThere are substantial local alterations in structure in the N-terminalω-loop of PT1 upon lysophosphatidylserine binding. The ω-loop wasoriginally defined as comprising residues 1–12 or so7. In our studies,using the definition of ω-loops proposed by Lesczczysnki and Rose21,we define the prothrombin ω-loop as comprising residues 1–7.

In our crystal structure of PT1 bound to Ca2+ ions the regionbetween Lys3 and Phe5 is flexible. Two alternate conformations of theω-loop predominate (Fig. 3c). In one conformer, ω-loop 1, the mole-cule adopts the conformation for Lys3, Gly4 and Phe5 observed bySoriano-Garcia et al7. The hydrophobic side chains of Phe5, Leu6 andVal9 are aligned with each other and more or less perpendicular to theplane formed by Ca2+ ions 1–6 (Fig. 3c, cyan). In the alternate confor-mation, ω-loop 2, the backbone shifts as much as 5.1 Å (at Gly4 Cα)

and the Phe5 ring is rotated so that it is essentially perpendicular to theside chains of Leu6 and Val9 (Fig. 3c, magenta). The position of theLys3 side chain cannot be determined because of weak electron densitycaused by high mobility. In the presence of lysophosphatidylserine thebackbone in the region around Lys3, Gly4 and Phe5 is all in the ω-loop2 conformation. Van der Waals and polar interactions between theglycerol backbone of lysophosphatidylserine and the backbone of Leu6and Gla7 stabilize the ω-loop. In the ternary complex the Lys3 sidechain is fixed in the presence of lysophosphatidylserine through inter-actions between the Nζ of Lys3 and the acyl and two phosphoesteroxygens of the lipid. The Phe5 side chain adopts a position distinctfrom either of the two predominant conformers observed in thebinary complex.

Studies with 15N-labeled PT(1–46) indicate that similar lipid-dependent conformational stabilization of the ω-loop region of thehuman prothrombin Gla domain occurs in solution. 1H-15N HSQCexperiments with PT(1–46) in the presence of Ca2+ show two amideresonances, NHF5 and NH′F5 (Fig. 3a), for the backbone NH of Phe5indicating two predominant conformers present in the N terminus.Addition of increasing concentrations of lysophosphatidylserine to15N-labeled PT(1–46) in the presence of Ca2+ shifts the equilibrium infavor of the NH′F5 conformer. At a stoichiometric equivalence oflysophosphatidylserine and PT(1–46), most of the PT(1–46) is presentas the NH′F5 conformer in solution, consistent with the stabilizationof a single conformer observed crystallographically (Fig. 3d). 1H-15NHSQC experiments with 15N-labeled PT(1–46) carried out in thepresence of small unilamellar vesicles composed of phosphatidyl-choline and phosphatidylserine (80:20) in the presence of Ca2+

demonstrate that binding to vesicles similarly shifts the conforma-tional equilibrium (data not shown).

DISCUSSIONPhosphatidylserine specificity of other Gla domainsThe structure of the ternary complex of PT1, lysophosphatidylserineand Ca2+ accommodates a body of experimental data describing prothrombin-phospholipid membrane interaction. Whereas otherphospholipid head groups can be accommodated spatially in the ser-ine head group–binding site, binding of a positively charged headgroup such as ethanolamine or choline is electrostatically unfavorable.

NATURE STRUCTURAL BIOLOGY VOLUME 10 NUMBER 9 SEPTEMBER 2003 753

a b c d

Figure 3 Comparison of the solution and crystal structures of prothrombin Gla domains. (a) HSQC spectrum showing the backbone and side chain 1H-15Nresonances of PT(1–46) taken at pH 7.4 and 5 °C. Phe5, Arg10 and Arg16 are 15N-labeled. (b) The region of the 1H-15N HSQC spectrum containing theNHε resonance of Arg10 is expanded. Spectra taken with no lysophosphatidylserine (black), 0.1 equivalents of lysophosphatidylserine (green), 0.5equivalents of lysophosphatidylserine (blue) and 1.0 equivalents of lysophosphatidylserine (red) are superimposed. The resonance of the NHε of Arg10 shiftsdownfield with increasing lysophosphatidylserine concentration. (c) Cα-trace from residues 1–25 of PT1 in complex with bound Ca2+ ions. The two highlyrepresented conformers in the N-terminal region of PT1 are in cyan and magenta. Bound Ca2+ ions 5 and 6 are black spheres. (d) The region of the 1H-15NHSQC spectrum containing the backbone amide resonances of Phe5 collected at stoichiometric equivalence of PT(1–46) and lysophosphatidylserine in thepresence of Ca2+ at pH 7.4 and 5 °C. The two backbone resonances arising from the two conformations observed for Phe5 are present but the majorresonance is now that of NH′F5.

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754 VOLUME 10 NUMBER 9 SEPTEMBER 2003 NATURE STRUCTURAL BIOLOGY

The phosphatidylserine specificity of prothrombin binding to phos-pholipid membranes is explained by the multiple interactions of theserine head group with Ca5 and Ca6 and Gla17 and Gla21. These lig-ands for lysophosphatidylserine are essential elements of a canonicalGla domain. Thus the structure predicts a similar specific serine headgroup–binding site in the Gla domains of other vitamin K–dependentproteins.

Arg16 is the only highly conserved cationic residue among Gladomains (human and bovine protein Z have a lysine). Interaction ofthis residue with a lysophosphatidylserine phosphate oxygen is likelypreserved in all Gla domains. Mutation of this arginine residue inhuman protein C decreased the binding affinity of protein C for phos-phatidylserine-containing phospholipids22. Differences in the bindingaffinity of Gla domain–containing proteins for phosphatidylserine-containing membranes likely depend on interactions of nonconservedGla domain residues with phosphatidylserine. Alternatively otherinteractions of the Gla domain with the membrane, for example inser-tion of ω-loop residues into the lipid bilayer, may affect affinity for themembrane.

Functional implications of the modelThe importance of negatively charged phosphatidylserine to vitaminK–dependent membrane binding suggests an ionic interaction.However, membrane binding of these proteins is quite insensitive toionic strength23. A body of evidence suggests that hydrophobicresidues of the ω-loop are important in membrane binding15−18 butchanges in membrane vesicle size or surface pressure expected to alteraffinity of membrane-inserted proteins show little effect on prothrom-bin membrane binding5. Our structural data help to resolve these contradictions. Binding of the prothrombin Gla domain to phos-phatidylserine-containing membranes is mediated by multiple typesof interactions: calcium coordination, ionic, van der Waals andhydrophobic. Interruption of one type of interaction, for examplecharge-charge interactions by manipulation of ionic strength, wouldbe unlikely to obliterate protein-membrane binding24.

The modest effect observed in mutational analyses of putativemembrane-interacting residues in the Gla domains of vitaminK–dependent proteins has been interpreted to suggest that the tar-geted residues are not involved in a primary phospholipid-bindingsite5,16. However, the large number of interactions between lysophos-

phatidylserine and Ca5, Ca6 and the N-terminal residues of PT1 sug-gests that mutations that affect any one of these contacts have modestimpact on overall prothrombin-phospholipid interaction.

Our structure is not in agreement with the observation that a con-formational transition of Pro22 from trans to cis is critical for pro-thrombin membrane binding25. In agreement with moleculardynamics calculations26, we observe the trans conformation for Pro22in both the PT1–Ca2+ and PT1–Ca2+–lysophosphatidylserine com-plexes. It has been reported that membrane-bound PT1 binds a greaternumber of Ca2+ ions than PT1 (refs. 27−29). We find the same numberof bound Ca2+ ions in the ternary and binary complexes. This may

Figure 4 Model of PT1 interacting with a phospholipid bilayer. PT1 is shownbound to one leaflet of a phospholipid bilayer composed ofphosphatidylcholine with a single lysophosphatidylserine molecule (shownas a stick model). The head group of the lysophosphatidylserine is bound toPT1 as in our crystal structure but the acyl chain of the lysopho-sphatidylserine is elongated and inserted into the lipid bilayer. The residuesof the Gla domain ‘hydrophobic patch’ of Ca2+-liganded PT1 are insertedinto the interstitial region of the lipid bilayer. PT1 is shown in a space-fillingmodel with residues of the ω-loop in yellow, the side chain nitrogens of Lys3,Arg10 and Arg16 in blue and Ca2+ ions in black. Lysophosphatidylserine:carbon, teal; oxygen, red; nitrogen, blue; phosphorous, green.

Table 1 Data collection and refinement statistics for PT1–Ca2+

and PT1–Ca2+–lysophosphatidylserine

Binary complex Ternary complex

Data Collection

Cell parameters (Å)

a 39.19 39.13

b 53.44 53.42

c 128.17 127.74

Space group P212121 P212121

Number of crystals used 1 1

Diffraction limit (Å) 1.90 2.3

Number of reflections

Total 150,545 112,656

Uniquea 21,658 (2,064) 11,566 (1,127)

Overall data redundancyb 7.0 9.7

Data completeness (%)a 98.3 (96.3) 92.2 (93.4)

Rmergea, c 0.077 (0.379) 0.085 (0.314)

I / σa 18.5 (2.6) 15.8 (2.8)

Refinement

Total atoms 1,314 1,270

Carbohydrate moieties 3 3

Resolution for refinement (Å) 32.0–1.90 41.0–2.30

Rcrysta, d 0.219 (0.281) 0.236 (0.281)

Rfreea, e 0.253 (0.315) 0.274 (0.312)

Average B-value (Å2) 29.0 46.1

Wilson B-value (Å2) 18.6 38.5

R.m.s. deviations

Bond length (Å) 0.019 0.006

Bond angle (°) 2.2 1.9

aValues in parentheses are for highest-resolution shell (1.97–1.90 Å for PT1–Ca2+ structure;2.38–2.30 Å for PT1–Ca2+–lysophosphatidylserine complex). bData redundancy is defined asnumber of observations per reflection. cRmerge = Σ|I – <I>| / ΣI, where I is the ith observation ofthe intensity of the hkl reflection and <I> is the mean intensity from multiple measurements ofthe hkl reflection. dRcryst = Σh||Fobs(h)| – |Fcalc(h)|| / Σh|Fobs(h)|, where |Fobs(h)| and |Fcalc(h)| arethe observed and calculated structure factor amplitudes for the hkl reflection, respectively.eRfree is calculated over reflections in a test set (10% of total reflections) not included in theatomic refinement.

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reflect differences in binding between soluble phospholipid and amembrane surface. A potential phospholipid head group–binding sitehas been identified in the surface cavity surrounded by Gla30, Gla8and Lys11 (ref. 30). This site is less accessible in the PT1–Ca2+–lysophosphatidylserine crystal than the site we have identified. Wecannot conclude whether or not this cavity is a second site forlysophosphatidylserine binding.

Comparison to other phosphoserine binding proteinsThe molecular interactions between a soluble phosphoserine-contain-ing compound and several other Ca2+-dependent membrane-bindingproteins have been elucidated: annexin V with glycerophosphoserineand the C2 domains of cytosolic phospholipase A2 with dicaproyl-phosphatidylserine31,32. The phosphatidylserine-binding regions ofannexin V and phospholipase A2 do not bear structural homologywith the Gla domains. Nevertheless, interactions of all of these pro-teins with membranes share some common features. Protein mem-brane binding is proposed to involve shallow penetration of a portionof the protein into the membrane bilayer facilitated by bridging of thephospholipid to the protein through a protein-bound Ca2+ ion. Theprotein ligands interacting with bridging Ca2+ ions vary. A single Ca2+

found in the C2 domain of phospholipase A2 binds to a single phos-phate oxygen of dicaproyl-phosphatidylserine. A phosphate oxygenand serine carboxyl oxygen of glycerophosphoserine each bind oneprotein-bound Ca2+ ion in annexin V bridging two adjacent metalion–binding sites. In the prothrombin Gla domain, the carboxyl oxy-gen atoms of the serine head group bind Ca5 and Ca6, bridging thesetwo metal ion–binding sites while basic side chains bind the phosphategroup. Finally, phosphoserine head group and glycerol backboneinteractions with specific protein ligands suggest specific binding sitesfor phosphatidylserine in each of these proteins.

A two-step mechanism has been proposed for binding of the C2domain of phospholipase A2 to the membrane surface. Ca2+

-dependent binding to negatively charged phospholipid at the interface is followed by specific protein-membrane interactions resulting in membrane penetration. A similar mechanism may be proposed for Gla domain–membrane binding. The Gla domain phosphatidylserine-binding site may promote optimal exposure of the ω-loop for penetration into the phospholipid membrane.Alternatively, when in the favorable conformation, the ω-loop mayinsert into the lipid bilayer positioning the phosphatidylserine headgroup–binding site in the optimal position for engaging its ligand.

Our model of membrane binding by the Gla domain–containing vit-amin K–dependent proteins (Fig. 4), with elements of both calciumbridging involving γ-carboxyglutamic acid and phosphoserine, and thehydrophobic mechanisms previously proposed for this interaction,provides a new paradigm for this class of interfacial membrane-bindingproteins. Salient features of this model include the presence of anappropriately charged pocket in the Gla domain for the phosphoserineof phosphatidylserine, electrostatic interaction of the carboxyl group ofthe serine head group with the γ-carboxyglutamic acid–calcium net-work of the Gla domain, the formation of salt bridges between the pos-itively charged arginine and lysine side chains with the phosphateoxygens, and the alignment of the fatty acid tail of lysophosphotidyl-serine with the hydrophobic patch of the Gla domain.

METHODSCrystallization and data collection. PT1 was purchased from HaematologicTechnologies and further purified using calcium-dependent phospholipidchromatography. Crystals of PT1 and Ca2+ ions were obtained by vapor diffu-sion from 20% w/v PEG 8000, 0.5 M NaCl, 10 mM CaCl2, 50 mM HEPES

(N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid), pH 7.6. Crystalswere soaked briefly in the crystallization solution containing 15% v/v glycerolimmediately before data collection. Final X-ray diffraction data for thePT1–Ca2+ crystal was collected on beamline X12C at Brookhaven NationalLaboratories (Upton, New York, USA). Lysophosphatidylserine (1-oleoyl-2-hydroxy-sn-glycerol-3-phospho-L-serine sodium salt, >99% purity) was pur-chased from Avanti Polar Lipids. A crystal of PT1 with bound Ca2+ ions, 0.3 ×0.3 × 0.4 mm, was soaked in 25% w/v PEG4K, 0.1 M HEPES, pH 7.5, 2.0 MCaCl2 (solution A) overnight, then in 1 mM lysophosphatidylserine in solutionA overnight and finally in solution A containing 1 mM lysophosphatidylserineand 2 mM CaCl2 for 3 h. The crystal was briefly (<1 s) dipped into a cryopro-tectant solution (solution A, 15% v/v glycerol) and immediately frozen at–160 °C before data collection. X-ray diffraction data was collected with an in-house 5 kW Cu X-ray power source focused with an Osmic confocal mirror to0.2 × 0.2 mm. The diffraction data was recorded on a Mar300 image plate. Allthe diffraction data were indexed and processed with DENZO33 and scaled withSCALEPACK33.

Structure determination and refinement. A molecular replacement method34

was used to fit the PT1 and Ca2+ ion model (PDB entry 2PF2) into our nativePT1–Ca2+ data. Model refinement and σA-weighted omit map generation35

were carried out by CNS version 0.9 (ref. 19). After rigid-body refinement ofthe PT1–Ca2+ model, electron density for the carbohydrate moieties bound toAsn77 and Asn101 was found in the σA-weighted 3Fo – 2Fc map and the carbo-hydrate moieties were built in with program O36. The model was optimizedfurther by iterative model minimization, solvent water search, temperaturevalue refinement and manual model building. Alternative conformations forthe main chain atoms of residues 3–5 and the side chains of Glu49 and Glu86were built in to yield the final model for PT1 and Ca2+ ions. The relative occupancies of the two conformations of residues 3–5 were estimated by theoccupancy refinement protocol in CNS. The structure of the PT1–Ca2+–lysophosphatidylserine complex was determined similarly to that of thePT1–Ca2+ complex except that our PT1–Ca2+ structure was the initial model.Electron density for lysophosphatidylserine was found from the 2Fo – Fc, σA-weighted map in the final stage of model building. A lysophosphatidylser-ine molecule was built and fitted this density well. Positional refinement andsimulated annealing protocols37 optimized the lysophosphatidylserine mole-cule position but did not shift it substantially. The temperature factor andoccupancy of lysophosphatidylserine were estimated by alternate cycles of tem-perature factor and occupancy refinements. The Ramachandran plot shows87.9% of residues in the most favored region, 10.5% in the additional allowedregion and 1.6% in the generously allowed region. For both the PT1–Ca2+ andPT1–Ca2+–lysophosphatidylserine structures, no notable electron density features were found in the final Fo – Fc residual maps.

PT(1–46) lysophosphatidylserine interaction by NMR. PT(1–46) peptideswere synthesized as described previously with the exception that 15N-labeledamino acids (phenylalanine (15N, 98%) and arginine (U-15N4, 98%);Cambridge Isotope Laboratories), were selectively incorporated at residuesPhe5, Arg10 and Arg16 (refs. 18,38). NMR samples contained 0.075 mM pep-tide, 2.25 mM CaCl2, pH 7.4, 90% H2O/10% D2O. All NMR spectra wereacquired at 5 °C on a Varian INOVA 500 MHz spectrometer. 2D 1H-15N-heteronuclear single-quantum correlation (HSQC) spectra were acquired withspectral widths of 8,000 Hz and 2,127 Hz for 1H and 15N, respectively, with2,048 real data points in t2 and 1,000 transients for each of the 64 increments int1 (ref. 39). Initial 1H-15N resonance assignments were made from a 2D 1H-15NTOCSY-HSQC40. 1H chemical shifts were referenced to the H2O resonance,which was calibrated at 278.5 K using 2,2-dimethyl-2-silapentane-5-sulfonicacid and calculated for 15N chemical shifts using the aforementioned 1H refer-ence and the gyromagnetic ratio of 0.101329118 (ref. 41). Lysophosphat-idylserine was titrated into the peptide sample to stoichiometric equivalencyusing a 0.67 mM stock. After the addition of each aliquot of lysophos-phatidylserine, the sample was sonicated on ice for 30 min. All spectra wereprocessed using FELIX2000 (Accelrys).

Generation of model. The Gla domain bound to lysophosphatidylserine asdetermined from our crystal structure was manually docked to a membrane

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surface with the ω-loop penetrating the lipid bilayer to the depth predicted byFalls et al18. The lysophosphatidylserine acyl chain was then manually reposi-tioned to insert into the lipid bilayer. No energy minimization protocol wasapplied to the model.

Coordinates. The atomic coordinates for PT1 bound to Ca2+ and PT1 bound toCa2+ and lysophosphatidylserine have been deposited in the Protein Data Bank(accession codes 1NL1 and 1NL2, respectively).

ACKNOWLEDGMENTSWe thank Brookhaven National Laboratories for time on beamlines X12C, X8Cand X25 and J. Wang for assistance with collection of X-ray data. This work wassupported by grants from the US National Institutes of Health to B.C.F., B.S., B.F.and M.A.G. and from the American Heart Association to A.C.R.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 27 February; accepted 24 July 2003Published online at http://www.nature.com/naturestructuralbiology/

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