The EMBO Journal vol.9 no. 1 2 pp.3867 - 3874, 1990

The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes Robert Huber, Jurgen Romisch' and

Eric-P.Paques' Max-Planck-Institut fur Biochemie, D-8033 Martinsried and 'Forschungslaboratorium der Behringwerke, D-3550 Marburg/Lahn, FRG Communicated by R.Huber

Human annexin V (PP4), a member of the family of calcium, membrane binding proteins, has been crystallized in the presence of calcium and analysed by crystallography by multiple isomorphic replacement at 3 A and preliminarily refmed at 2.5 A resolution. The molecule has dimensions of 64 x 40 x 30 A3 and is folded into four domains of similar structure. Each domain consists of five a-helices wound into a righthanded superhelix yielding a globular structure of 18 A diameter. The domains have hydrophobic cores whose amino acid sequences are conserved between the domains and within the annexin family of proteins. The four domains are folded into an almost planar array by tight (hydrophobic) pair-wise packing of domains II and III and I and IV to generate modules (II-III) and (I-IV), respectively. The assembly is symmetric with three parallel approximate diads relating II to Ill, I to IV and the module (H-IIl) to (I-IV), respectively. The latter diad marks a channel through the centre of the molecule coated with charged amino acid residues. The protein has structural features of channel forming membrane proteins and a polar surface characteristic of soluble proteins. It is a member of the third class of amphipathic proteins different from soluble and membrane proteins. Key words: calcium channel/crystal/membrane protein/ protein/structure

Introduction Annexins are a class of widely distributed proteins which bind to phospholipids and membranes in a Ca2+ dependent manner. They are found in several cell types and in many species (see Geisow and Walker, 1986; Crompton et al., 1988; Klee, 1988; Smith et al., 1990 for reviews). Their intracellular localization and association with cytoskeletal proteins suggest participation in cytodynamic events, like membrane fusion and exocytosis. It has also been proposed that annexins are involved in the cell signalling pathways (Creutz et al., 1987; Burgoyne and Geisow, 1989; Ross et al., 1990). The synthesis of annexin I is inducible by glucocorticoids in some cell types (Hirata et al., 1980; Mitchell et al., 1988; Goulding et al., 1990), but its relevance is a matter of controversy (Hullin et al., 1989; Isacke et al., 1989). Anticoagulant and anti-inflammatory properties have been Oxford University Press

attributed to annexins (Reutelingsperger et al., 1985; Tait al., 1988; Romisch and Heinburger, 1990). By binding to cell membranes they are thought to inhibit the association of blood coagulation factors with cell surfaces and to prevent their activation. A possible antithrombotic function of annexins remains to be demonstrated, as their extracellular concentrations are extremely low (Flaherty et al., 1990; et

Goulding

et

al., 1990).

Annexins associated with the plasma membrane intracellularly inhibit degradation of membrane lipids by phospholipase, especially PLA2, which releases the inflammation mediator arachidonic acid. Anticoagulant and anti-inflammatory properties are mediated by calcium. The annexin family includes eight unique proteins of known amino acid sequences. Amino acid sequences of the molecules discussed here were taken from the MIPSX database and compared. These proteins are: human annexin V (Grundmann et al., 1988); mouse annexin II (Saris et al., 1986); human annexin VI (Crompton et al., 1988b; Moss et al., 1988) (see Smith et al., 1990 for review). Their functional properties, affinity and specificity for lipids and membranes differ vastly (Tait et al., 1988). One of the most effective anticoagulant annexins is known as placenta protein 4 (PP4) (Tait et al., 1988, 1989; Romisch and Heimburger, 1990). Its cDNA encodes an unglycosylated protein of 320 amino acids with a molecular mass of 35 935 daltons (Grundmann et al., 1988). PP4 is identical to the 'vascular anticoagulant' (VAC-alpha) (Maurer-Fogy et al., 1988) also called 'inhibitor of blood coagulation' (IBC) (Iwasaki et al., 1987) and 'endonexin II' (Kaplan et al., 1988). The in vitro anticoagulant and antiinflammatory properties of this protein have been intensively investigated (Tait et al., 1988; Romisch and Heimburger,

1990).

Little is known about the structure of annexins and the molecular basis of their calcium mediated association with membranes. Preliminary structural studies of twodimensional crystals of lipid monolayer bound annexin VI (p68) by electron microscopy, revealed a trimeric arrangement of molecules resolved in six protein domains (Newman et al., 1989). Three-dimensional crystals of annexin VI (human) (Newman et al., 1989) and annexin V (human, rat) (Lewit-Bentley and Bodo, 1989; Seaton et al., 1990) have been obtained, but no structure reported. We describe here the preparation, crystallization and the molecular model of anticoagulant human placenta annexin V and attempt to relate this to its functional properties. This work was stimulated by our programme of structural studies of proteins involved in blood coagulation (Bode et al., 1989; Rydel et al., 1990) and by our continuing interest in membrane protein structures (Deisenhofer et al., 1985). We confirm that annexin V consists of four tandem repeats of similar structure. The domains exhibit a novel type of helix assembly. The four domains are folded into a highly symmetrical molecule. The secondary structures of the 3867

R.Huber, J.Romisch and E.-P.Paques

./

Fig. 1. Electron density and model around residue Trpl87 of the averaged MIR map at 3 A resolution (a), and of the phase combined map at 2.6 A resolution (b).

domains were correctly predicted from the amino acid sequence but the suggested tertiary fold is wrong (Taylor and Geisow, 1987). We follow the suggested common terminology for annexins (Klee, 1988; Crumpton and Dedman, 1990; Smith et al., 1990).

Molecular structure The polypeptide chain is folded into four domains consistent with the amino acid sequence repeats (Grundman et al., 1988). The domains are bounded as follows: tail, 5-16; domain I, 17-88; domain II, 89-159; connector, 160-167; domain HI, 168-246; domain IV, 247-317. The N-terminal tail runs along the surface of domain I and crosses over to domain IV, where it is bound non-covalently close to the C terminus. It acts as a clamp helping to link domains I and IV and to generate a cyclic arrangement of the domains. Similarly, the segment which covalently links domains II and III is extended. The connections between domains (I,H) and (Ill,IV) are short, best described as sharp kinks between the C- and N-terminal ca-helices of contiguous domains.

3868

Fig. 2. Scheme of the domain arrangement and dimensions of annexin V as seen from the concave side of the molecule. Domains I and IV and II and III, respectively, form two tightly associated modules which interact more loosely. The contacts around local diads B and C are largely apolar, while contact A is polar, generating a channel along Al The lines indicate the connecting segments which are long between domains (II,III) and (I,IV) (non-covalent) and short between I and III and IV.

Human annexin V structure

a

b IEIB Iv A

III

Cj~~~~~~ii

rvc

III15

N

Fig. 3. (a) Helix cylinder plot of annexin V as seen from the side of the convex surface. The four domains are indicated I, II, III and IV and the helices A-E. (b) Side view of annexin V.

An annexin domain is cube-shaped with a side length of 18 A (Figures 2 and 3). Its polypeptide chain is folded in five a-helices in accord with secondary structure predictions (Taylor and Geisow, 1987). The helices are: IA 17-29, IB 34-45, IC 46-60, ID 65-74, IE 75-86; IIA 88-102, IIB 106-117, IIC 118-134, IID 136-145, IIE 147-158; IIIA 169-186, IIIB 190-201, IIIC 203-218, IIID 220-226, IIIE 231-246; IVA 247-259, IVB 266-277, IVC 280-292, IVD 295-305, IVE 306-3 17. The five helices within each domain are wound into just less than two turns of a right-handed superhelix. An alternative description of the fold is a four-helix bundle with helix C crossing over and linking the helix-turn-helix substructures, A -B and D - E, respectively (Figures 3 and 4). The connectivity is thus different from the 'E - F hand' calcium binding proteins (Tufty and Kretsinger, 1975; Herzberg and James, 1985; Szebenyi et al., 1986; Declerc et al., 1988) and other four a-helical proteins (see Weber and Salemme, 1980 for a review) which are composed of cyclically joined a-helices. This latter arrangement is 2-fold symmetric around the bundle axis, while the annexin domain has translational symmetry between the A-B and D-E helix pairs. There is to our knowledge also no obvious relationship to other a-helical proteins (interleukin 2: Brandhuber et al., 1987; colicin A: Parker et al., 1989) or substructures of large a-helical enzymes (citrate synthase: Remington et al., 1982). All four annexin domains are similar and superimpose closely. A conspicuous difference concerns helix IIID which is shorter than in the other domains, probably as three consecutive charged residues Asp226, Arg227 and Glu228 cannot be accommodated in helical geometry. Although of small size the domains have hydrophobic cores shown in Figure Sa. The four domains are assembled as indicated in Figure 2 with molecular dimensions of 64 x 40 x 30 A. Three local diad axes are generated, two of which relate domains I with IV (B) and III with 11 (C), respectively. These non-covalent -

interdomain contacts are tight and mediated by hydrophobic residues (see Figure Sa) thus generating the modules (I -IV) and (II-IIl), respectively. The two modules interact less tightly mostly by polar charged contact residues (Figure Sb), thereby forming a unique channel along the central diad A described below. An alternative description of the helix assembly of the entire molecule is four four-helix rows, which are closely spaced within the modules and more distant between them (Figure 3a). The helices within the rows are mixed, antiparallel (A - B, D - E, within domains) and parallel (B - E, between domains). The four connecting helices C lie flat between the closely spaced helix rows. The side view of Figure 3b shows the slightly curved plane formed by the domains. We distinguish a concave underside where the N terminus is located and a convex upper side. The framework of the channel is formed by helices IIA, IIB, IVA, IVB and coated with charged residues listed in ascending order from the concave surface: Glu 120, Glu12 1, Arg276, Asp280, Asp92, Arg117, Glu95, Glul12, Arg271, LyslO8, Lys79, Asp265. It is bounded by Tyr91, 94 and Tyr250, 257. All of these side chains have defined electron density and are well ordered. An ion would move through the channel requiring slight side chain rearrangements. A particular impediment are the salt bridges formed by Asp280 -Arg276, Asp92 -Argi 17 and Glu 112 -Arg271 (in bold above). These residues are invariant in annexins (see alignment Figure 3 in Lee Burns et al., 1989). The molecular structure thus has elements expected for a channel forming membrane protein: an assembly of almost parallel helices around a central channel. However, annexins are water soluble and have polar residues in the periphery (Figure Sb). Annexins do indeed interact with membranes in a calcium dependent manner and change membrane properties. Annexin VI, for example, induces an increase in surface pressure of monolayers of phosphatidylethanolamine (Newman et al., 1989), but smaller than expected for complete penetration of the protein in the lipid monolayer. 3869

R.Huber, J.Romisch and E.-P.Paques

Fig. 4. Line drawing connecting the C' atoms seen along the local diad axes. Domains I and II are drawn with continuous lines, Ill and IV are dotted. The view is from the convex side.

Human synexin (annexin VI) shows calcium channel activity (Lee Burns et al., 1989). Synexin has a C-terminal region of 299 amino acids highly homologous to annexin V and a highly hydrophobic N-terminal part of 167 residues very rich in glycines, prolines and aromatic residues. The latter might be involved in membrane interaction but does not have features of an ion selective channel. The C-terminal 15 residues of the N-terminal region are homologous to the tail of annexin V suggesting a similar apposition at the concave side of the core region. We suggest therefore that the central channel seen in annexin V represents the selective, voltagegated calcium channel in synexin. As most of the charged amino acids coating the inner wall of the channel are highly conserved in all known annexins we expect common similar channel characteristics.

Implications for other annexins Eight unique proteins have been defined by sequence data to belong to the annexin family. Annexins I-V and VII are organized in tetrad repeats, whereas annexin VI has eight repeats (see Smith et al., 1990). The amino acid sequence conservation between the family members is mostly substantially higher than 40% identity suggesting close structural relationship to annexin V. In particular the residues forming inter-helix loops, the hydrophobic cores of the domain and the tight inter-domain contacts are conserved. A consensus sequence was recognized in annexin domains of - 15 amino acid residues, beginning with residues 28, 100, 185, 259 in domains I-IV of annexin V (Geisow et al., 1986; Klee, 1988). These segments make up the loop between helices A and B and most of helix B in each domain. The loops are very exposed on the convex surface of the molecule. An antibody directed against the loop region (26-35 in domain I) recognizes all annexins (Kaetzel and Dedman, 1989). Helix B is central in the tight contact between the diad related domains (I-IV) and (II-111), 3870

respectively. The hydrophobic stretch of residues within the consensus sequence mediates this contact. Annexin VI (p68) has an octad repeat (Moss et al., 1988). Sequence alignment indicates duplication of a tetrad repeat (Crompton et al., 1988a). Various reconstructions on the basis of the structure of annexin V may be considered, but in view of the modular, symmetric construction of annexin V a lateral, planar expansion by addition of two more twodomain modules is most likely. Electron microscopy has suggested a cylindrical molecule with dimensions of 100 A by 35 A diameter (Newman et al., 1989) in accord with this suggestion if some stain penetration is taken into account. A wealth of data have been accumulated for annexin II, which may be correlated with the structure of annexin V. Annexin II (p36, calpactin I) is isolated as a heterodimer with a small protein component, p11 (p36-p 1 )2. It was shown that the interaction between p36 and pl1 is mediated through the N-terminal 12 residues of annexin II which have no counterpart in annexin V as it is shorter by 18 residues. The N terminus is protease sensitive and cleaved by trypsin and chymotrypsin at residues 10 and 12 (annexin V numbering, Johnsson et al., 1988). Cleavage occurs within the extended segment of the N terminus (Figure 4). It bears tyrosine and serine/threonine phosphorylation sites (Johnsson et al., 1988). Epitope mapping has shown that in annexin H the peptide segments around 25 and 65 belong to the same (discontinuous) epitope (Johnsson et al., 1988b). These residues are homologous to 7 and 47 in annexin V which are spatially close enough (14 A) to form a common epitope. In experiments of limited proteolysis of annexin II cleavage occurs between residues homologous to Lys 186 -Trp 187 in annexin V yielding two fragments of 20 kd and 15 kd (Johnsson and Weber, 1990a). These dissociate under denaturing conditions and reassociate when the denaturant is removed. Proteolysis occurs between helices IIIA and IIB in the exposed peripheral loop (Figure 4). The fragments may aggregate spontaneously driven by the formation of favourable tight II-III and I-IV contacts.

Human annexin V structure

a

b

Fig. 5. C' line drawing with hydrophobic (a) and charged amino acid side chains included (b). The view is from the convex side.

Implications for calcium and phospholipid binding The annexin V crystals were produced and kept in the presence of > 1 mM calcium which should be sufficient to saturate the calcium sites. However, calcium binding may be weakened by the high concentration of ammonium sulphate in which the crystals are stabilized. Two observations support the notion that the molecular structure observed and described is the calcium liganded form: the protein fails to crystallize in the absence of calcium under otherwise identical conditions and crystals of both forms remain unchanged at much higher calcium concentrations than used for crystallization. In the electron density map we have so far been unable to identify unequivocally bound calcium and conclude that the calcium sites are not

completely occupied or are disordered under the experimental conditions. Prior to appropriate crystallographic experiments we refer to some pertinent biochemical observations made with the homologous annexin H (p36). A terbium and, by implication, calcium site has been located in annexin H and found to be within 8 A of the single tryptophan residue (Marriott et al., 1990). The tryptophan is at position 213 homologous to Phe 194 in annexin V. Annexin V also has a single tiyptophan at 187 in the turn between helices IlA-IHIB and located in a deep niche at the convex surface of the molecule. Phel94 is in Van der Waals contact with Trpl87. Acidic residues as possible calcium ligands in this area are 175, 190, 191, 192, 222 and 234. Most have well defined electron density, are ordered, but not paired in a geometry required for calcium sequestration. 3871

R.Huber, J.Romisch and E.-P.Paques Table I. Statistics of diffraction data and isomorphous replacement Derivative

Native MEHG HGNO TEME PTI6 CPBC

MEHG: HGNO: TEME: PTI6: CPBC:

Completeness

Measurements Total

Independent

212211 111192 97435 88089 94121 66495

29633 28748 20956 20679 17951 15462

0.70 0.92 0.87 0.97 0.81 0.83

0.89 0.94 0.93 0.98 0.91 0.80

RM

RFM

0.123 0.130 0.176 0.128 0.154 0.167

0.055 0.074 0.076 0.079 0.100 0.078

0.5 mg/ml CH3HgCl, 15 h soak 4.6 mg/ml K3Hg(NO2)4NO3, 50 h 1.7 mg/m] C(HgCH3COO)4, 50 h K2PtI6, saturated, 12 h 5.0 mg/mi (CH3)3PbCl, 15 days

Completeness of data above 2.5 a: first column co -2.3 A for native and 00-3.0 A for derivatives; second column 2.37-2.30 A for native and 3.1-3.0 A for derivatives

RM:

E(I - )/EI, for all

RFM:

for

averaged

measurements

Friedel pairs

Transformation of molecule A to B 0.99780 0.06618 Matrix: 0.99752 -0.06606 0.00542 -0.02407 62.90554 Vector: -57.22413

(orthogonal coordinates): 0.00382 -0.02437 -0.99970 153.69000

Heavy atom parameters X 0 MEHG FH/Res 1.64 116.109 0.1190 103.570 0.8293 12.100 0.7240 1.30 HGNO 98.898 0.1193 88.423 0.8295 19.247 0.7231 11.372 0.8275 1.56 TEME 78.282 0.1208 67.744 0.8281 139.310 0. 1601 98.416 0.1570 1.37 PTI6 131.106 0.8072 202.274 0.9197 31.866 0.3956 43.929 0.8059 120.791 0.0383 27.439 0.7324 0.94 CPBC 81.211 0.3695 45.545 0.4117

y

B1l

z

B13

B12

B22

B23

B33 0. 12E-02 0.96E -03

0.13E-02 0.84E-03 0. 13E-03

0.20E-03 -0.30E-03 -0.12E-02

0.13E-02 0.10E-02 0.98E-02

-0.11E-03 -0.57E-04 0.48E-02

-0.10E-02

0.75E-03 0. 16E-02 0.68E -03 -0.26E-02

0.35E-03 -0.12E-03 -0.12E-02 -0.23E-02

0.10E-02 0. 16E-02 0.88E-02 -0.21E-02

0.45E-04 -0.38E-04 0.40E-02 0. 16E-02

0.10E-02 0.72E-03 -0.66E-03 0.89E -02

0.91E-03 0.86E -03 0.74E -02 0.73E-02

0.88E-03

0.10E-02 0.83E-02

0.31E-03 -0.73E-04 -0.24E-02

0. 14E-03 0. 15E-03

-0.50E-02

0.97E-02

-0.40E -02

0.10E-02 0.78E-03 0.12E-01 0.93E-02

0.52E-03 0.67E-03 0.46E-02 0.42E-02

0.4136 0.2689 0.4070 0.4114 -0.0001 0.2780

0. 17E-02 0.81E-02 0.75E-03 0. 13E-02 0.56E-02 0.29E-02

0.72E-03 0.13E-01 0.48E-05 0. 14E-02 -0.10E-02 0.15E-02

0.24E-02 -0.47E-02 0.26E-03 0. 16E-02 0.83E-03 0.47E-03

0.37E-02 0.10E-01 0.57E-03 0.33E-02 0.45E-02 0.63E-03

-0.28E-03 -0.33E-03 -0.47E-03 -0.12E-02 0.61E-02 0. 14E-03

0. 19E-02 0.34E-02 -0.23E-03 0.24E-03 0.59E-02

0.3944 0.3708

-0.27E-04 -0.10E-02

0.41E-03 -0.76E-03

-0.14E-03 0.42E-03

0.18E-03 -0.68E-03

-0.89E-04 0.29E-03

0.70E-03 0.16E-02

0. 14E-02

0.3943 0.0130 0.1097

0.0000 0.1880 0.4667

0.3948 0.0124 0.1132 0.0117

0.0004 0.1886 0.4681 0.1819

-0.16E-02

0.3953 0.0115 0.1747 0.4177

0.0002 0.1896 0.1786 0.0103

0.1425 0.0377 0.4712 0.1135 0.0248 0.1400 0.4296 0.0451

0.11E-02 0.52E-02 0.10E-02 0. 15E-02

0.55E-02

-0.34E-02

0.12E-03

Figure of merit oc -3 A: 0.62 0: occupancy in relative units. X,Y,X: fractional coordinates. anisotropic temperature parameters. B1I -B33:mean heavy atom contribution/residual. FH/Res:

Other findings indicate a direct or indirect involvement of the N terminus in calcium and/or phospholipid binding: annexin II (p36) may be phosphorylated by src tyrosine protein kinase at Tyr24 (Johnsson et al., 1986, 1988a).

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The phosphorylated species has a lower affinity for phosphatidylserine (Powell and Glenney, 1987). Although annexin II is longer at the N terminus by 18 residues compared with annexin V the common portion is clearly

Human annexin V structure

homologous. In particular the segment LRGTVTDFPGF (5-15) reads AYGSVVAYTNF (23 -34) in annexin II and contains the phosphorylated tyrosine. This segment interacts

with domains I and IV in annexin V and is likely to adopt similar conformation and binding site in annexin II. LRGT is located exactly at the entrance to the central channel at the concave side. Conversely p 11 binding to the N-terminal segment decreases the calcium requirement for phospholipid binding (Johnsson and Weber, 1990a,b). p11 binds to the first 12 residues of annexin H (Johnsson et al., 1988a) which have no counterpart in annexin V but must be located at the concave side of the molecule restricting the location of p 11 to the same area. Proteolytic cleavage of the tail has an effect similar to p 11 binding and reduces the calcium requirement for phospholipid binding (Drust and Creutz, 1988; Ando et al., 1989). We propose that the N-terminal segment modulates calcium-phospholipid binding essentially by tighter or looser interaction with the central channel in a way which remains to be defined in detail. The finding of a calcium channel in annexin VII and its structural definition annexin V suggests that it harbours also calcium binding sites. The location of p 1 at the concave side of the annexin core implies membrane attachment to the opposite convex side, but details remain to be established. Calcium and phospholipid binding in annexins are linked. Chelation of the lipid phosphoryl/carboxyl groups and protein groups by calcium has been suggested (Taylor and Geisow, 1987). The presence of a central channel with calcium activity (in annexin VII) suggests an allosteric linkage in addition whereby calcium stabilizes the closed circular domain assembly which might be the membrane active conformer. a

Annexins, amphipathic proteins Annexins are water soluble and membrane binding proteins. Annexin V has abundant polar, charged amino acid residues on its surface like soluble proteins. However, annexins interact strongly with membranes in the presence of calcium and exhibit properties of proteins integrated in the membrane: increase in surface pressure of phospholipid layers and channel activity. Two possible explanations offer themselves: a profound structural rearrangement of the protein ('inside-out') or rearrangement of the membrane. The former has been proposed for colicin A (Parker et al., 1989) but seems unlikely for annexins which show in water structural features expected for channel forming membrane proteins. The latter explanation seems more plausible for annexins and we propose that rearrangement of the membrane bilayer phase allows protein integration such that the phospholipid head groups interact with the polar protein surface possibly mediated by calcium ('inverted micelle'). It is well known that acidic phospholipids in the presence of calcium tend to form a hexagonal phase with inverted micelles as building blocks (see Gennis, 1989 for review).

Materials and methods

Crystallization and structure analysis The protein was dissolved in 3 mM MOPS buffer, pH 7.0, to a concentration of 9 mg/ml. Droplets were made from 10 A1 protein solution, 1 Al 20 mM CaCI2 solution and 1.5 I precipitating buffer were added. Crystallization was achieved at room temperature by vapour diffusion against 2.1 M of ammonium sulphate, 0.1 M Tris-chloride, pH 8.5. Crystals grew as pointed hexagonal columns and rhombohedra, side by side and intertwined. They were harvested into 3 M ammonium sulphate buffer, 0.1 M Tris-chloride, pH 8.5, 1 mM CaCI2. Both crystal forms diffract to high resolution and the hexagonal crystals were analysed in detail. The space group of the hexagonal crystals is P63 with lattice constant a = b = 98.6 A, c = 129.6 A, CY = 3 = 900, -y = 120'. The asymmetric unit contains two molecules. The rhombohedral crystals have space group R3 with cell constants (hexagonal setting) a = b = 99.6 A, c = 97.2 A, a = 3 = 90°, -y = 1200 and contain probably one molecule in the asymmetric unit. Although the hexagonal and rhombohedral crystals have very similar a and b axes, the interpenetration twins do not have coincident c axes. All X-ray experiments were conducted with a FAST television area detector system (Enraf Nonius, Delft) and a Rigaku rotating mode X-ray generator operated at 5.4 kW with an apparent 0.3 x 0.3 focal spot. X-ray intensities were evaluated with the MADNES system (Messerschmidt and Pflugrath, 1987) and scaled, absorption corrected and averaged (Messerschmidt et al., 1990). Data collection statistics are given in Table I. Heavy atom derivatives were prepared by soaking under the conditions given in Table I and were analysed by difference Patterson methods. Methyl mercurichloride, the first derivative made, was easily interpreted with two binding sites. The correct hand of the heavy atom structure was determined by single isomorphous replacement and solvent flattening. Other derivatives were comparably easily interpreted and included in phase calculation and parameter refinement. A Fourier map calculated at 3 A resolution allowed the tracing of the polypeptide chain and was further removed by averaging the densities of the two independent molecules. The symmetry relation between them was determined by picking 9.245 density points from the molecular area of molecule A and searching the optimal correlation with the electron density in the area of molecule B by alternate three-dimensional rotation and translation grid searches. The correlation increased from 290 to 508 (relative units) by shifting the molecule by a few tenths of 1 A and reorientation by -2° from a starting position obtained by visual examination. The transformation between molecules A and B is given in Table I. It corresponds to an almost exactly 2-fold rotation around an axis lying in the x y plane at an angle of 46.900 to the (orthogonal) y axis. The rotation angle is 178.8° with screw component 0.14 A. A model of the polypeptide chain was built for residues 5-317, with the interactive graphics program FRODO (Jones, 1978). Residues 1-4 and 318-320 have no defined electron density. This model was refined using EREF (Jack and Levitt, 1978) whereby the R-value was reduced from 0.40 to 0.25 from 8 to 2.5 A resolution without rebuilding. Interpretation is based on this preliminarily refined model. Figure 1 shows part of the electron density map and model around residue Trpl87 of the isomorphous and averaged (a) and combined map (b) where isomorphous phases have been combined with those calculated from the current model.

Acknowledgements We thank M.Schneider and I.Mayr for their excellent technical assistance with the computations and the crystallization, respectively. Dr M.Stubbs helped with suggestions on scientific and linguistic matters.

References Ando,Y, Imamura,S., Hong,Y.M., Owada,M.K., Kakunaga,T. and Kannagi,R. (1989) J. Biol. Chem., 264, 6948-6955. Bode,W., Mayr,I., Baumann,U., Huber,R., Stone,St R. and Hofsteenge,J. (1989) EMBO J., 8, 3467-3475. Brandhuber,B.J., Boone,T., Kenney,C. and McKay,D.B. (1987) Science, 238, 1707-1709. Burgoyne,R.D. and Geisow,M.J. (1989) Cell Calcium, 10, 1-10. Creutz,C.E., Zaks,W.J., Hamman,H.C., Crane,S., Martin,W.H., Gould,K.L., Oddie,K.M. and Parsons,S.J. (1987) J. Bio. Chem., 262, 1860-1868.

Isolation of annexin V Annexin V was isolated as described by Romisch and Heimburger (1990). Briefly, freeze-dried human placenta was extracted with citrate-containing buffer. After removal of the cell debris annexin V was purified by chromatography on phenyl, (calcium) heparin and Q-Sepharose, yielding a >95% pure preparation, which was free from other annexins.

Crompton,M.R., Moss,S.E. and Crumpton,M.J. (1988a) Cell, 55, 1-3.

Crompton,M.R., Owens,R.J., Totty,N.F., Moss,S.E., Waterfield,M.D.

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