Crystal and molecular structure of human annexin V after refinement

J. Mol. Biol. (1992) 223, 683-704

Crystal

and Molecular Structure of Human after Refinement

Implications

Annexin

V

for Structure, Membrane Binding and Ion Channel Formation of the Annexin Family of Proteins

Robert Huber, Robert Berendes, Alexander Burger Monika Schneider, Andrej Karshikov, Hartmut Luecke Max-Plan&-Institut fiir Biochemie Am Klopferspitz 18a, D-8033 Martinsried, Germany

Jiirgen

Riimisch

and Eric Paques

Forschungslaboratorien der Behringwerke D-3550 MarburglLahn, Germany (Received 18 July

1991; accepted 16 October 1991)

Two crystal forms (P6, and R3) of human annexin V have been crystallographically refined at 2.3 A and 2.0 A resolution to R-values of 0.184 and 0.174, respectively, applying very tight stereochemical restraints with deviations from ideal geometry of 001 A and 2”. The three independent molecules (2 in P63, 1 in R3) are similar, with deviations in Ca positions of 0.6 A. The polypeptide chain of 320 amino acid residues is folded into a planar cyclic arrangement of four repeats. The repeats have similar structures of five a-helical segments wound into a right-handed compact superhelix. Three calcium ion sites in repeats I, II and IV and two lanthanum ion sites in repeat I have been found in the R3 crystals. They are located at the convex face of the molecule opposite the N terminus. Repeat III has a different conformation at this site and no calcium bound. The calcium sites are similar to the phospholipase A, calcium-binding site, suggesting analogy also in phospholipid interaction. The center of the molecule is formed by a channel of polar charged residues, which also harbors a chain of ordered water molecules conserved in the different crystal forms. Comparison with amino acid sequences of other annexins shows a high degree of similarity between them. Long insertions are found only at the N termini. Most conserved are the residues forming the metal-binding sites and the polar channel. Annexins V and VII form voltage-gated calcium ion channels when bound to membranes in vitro. We suggest that annexins bind with their convex face to membranes, causing local disorder and permeability of the phospholipid bilayers. Annexins are Janus-faced proteins that face phospholipid and water and mediate calcium transport. Keywords:

crystal

structure;

annexin;

membrane

1. Introduction

protein;

calcium;

voltage-gated

channel

(Smallwood et al., 1990). More than ten members of the annexin gene family have been found and characterized (for a review, see Johnston & Siidhoff, 1990). Annexins have anticoagulatory and antiinflammatory properties possibly by interfering with membrane attachment of coagulation factors and phospholipase A,, respectively. Other intracellular functions proposed include membrane fusion, mediation of membrane cytoskeleton inter-

Annexins are cytosolic calcium, membrane-binding proteins widely distributed in different species and diverse tissues and cell types. Previously only studied in vertebrates, annexins have recently also been found in multicellular invertebrates, in Clrosophila (Johnston et al., 1990) and Dictyostelium (Gerke, 1991; Diiring et al., 1991) and in plants 683 0022%%336/92/030683-22

$03.00/O

0

1992

Academic

Press

Limited

684

R. Huber

action and, as found quite recently for lipocortin III (annexin III), hydrolysis of inositol-1,2-cyclic phosphate (Ross et aZ., 1990). Annexins I and II are phosphorylated by epidermal growth factor kinase, protein kmase C and the kinase encoded by Rous sarcoma virus oncogene, respectively (Gould et al., 1986; Johnsson et al., 1986; Schlaepfer & Haigler, 1987; for reviews, see Klee, 1988; Crompton et al., 1988a). A common property of the members of the gene family of annexins is their binding to negatively charged phospholipids in a caleium-dependent manner (for a review, see Johnston & Siidhoff, 1990). Despite diverse biological properties of members of the annexin family, their amino acid sequencesshow a high degree of identity. They have usually four, in annexin VI eight, tandem repeats of about 80 residues’ length, which are well conserved within the annexin family, and a more variable N-terminal segment (for reviews, see Geisow & Walker, 1986; Crompton et al., 1988a; Klee, 1988; Smith et aE., 1990). Annexins are amphipathic proteins distinct from soluble and integral membrane proteins. They are readily soluble in water and interact with membranes in a calcium-dependent manner. Some members also form voltage-gated calcium-specific channels when associated with membranes, a property of integral membrane proteins (for a review, see Pollard et al., 1990). We studied annexin V; a potent, anticoagulant from human placenta, by crystallography initially within our program of structural studies of coagulation factors (Bode et al., 1989; Rydel et al., 1990) and presented the crystal structure and molecular model of hexagonal and rhombohedral form of annexin V (Huber et al., 1990a,b). We showed that the molecule of 320 amino acid residues is almost entirely a-helical. The four tandem repeats are similarly folded into compact domains consisting of five a-helices wound into a right-handed super-helix. Four helices A, B, D and E have their axes approximately (anti-parallel, whereas the connecting helix C lies flat. The four domains are arranged in an almost planar, cyclic array such that domains II and III and I and IV, respectively, form tight modules with approximate 2-fold symmetry. A third local dyad (C) relates modules (II and III) and (I and IV), so that all four domains have their molecular axes as defined by the axes of helices A, B, D and E similarly oriented. Dyad C marks the center of the molecule and a very prominent hydrophilic pore, which we associated with the calcium selective channel found in annexin VII and V (Burns et al., 1989; Pollard et aE., 1990). In the rhombohedral crystal form we found three almost fully occupied calcium-binding sites formed by protruding polypeptide loops in homologous segments of repeats I, II and IV and two lanthanum-binding sites at repeat I. These metalbinding sites are all located at the convex face of the molecule suggested to be the membrane contact area. A study of annexin V bound to phospholipid

et al.

layers by electron microscopy and its comparison with the crystal structure did not indicate significant structural rearrangements (Mosser et aZ., 1991; Brisson et al., I991), justifying the interpretation of the functional properties of the membrane protein complex on the basis of the high resolution crystal structure. We describe here the crystal and molecular structure of human annexin V after refinement of the hexagonal and rhombohedral crystal forms and relate it to that of other annexins. A model for the annexin membrane complex is proposed and its calcium conduction and transmembrane voltage regulation properties discussed. 2. Experimental Methods (a) Crystallography The crystallization, data collection, phase determination, and preliminary refinement, has been described for both crystal forms (Huber et al., 1990a;b). Refinement was continued to convergence and is summarized in Table 1. The hexagonal and rhombohedral cryst,al form have R-values of 0184 from 8 to 2-3 A resolution (1 A = 91 nm) and O-174 from 8 to 2.0 A, respectively. Very tight stereochemical restraints were maintained throughout the refinement process, which was carried out in cycles of model building and model correction (using the program FRODO: Jones, 1978) and EREF (energy restrained) crystallographic refinement (Jack & Levitt, 1978). In the hexagonal crystal form 4 sulfate groups with half occupation but no calcium were found. The rhombohedral form has 3 calcium ions with occupancy 075 (Cal to Ca3), 2 calcium ions with occupancy 05 (Ca4 and Ca5) and 2 half occupied sulfate groups. The Ca4, Ca5 sites bind lanthanum strongly (Huber et al., 1990b). Main-chain segments with breaks or undefined electron density when contoured at @9 d are few: in R3, only the N and C termini 1 to 2 and 319 to 320 and residues 228 to 229. In P6,, only the N termini residues 1 to 2 for both molecules A and B. The C terminus is undefined from 319 in molecule A and there is a break at residue 318 in molecule B. There are breaks in the electron density of molecule B at position 31 to 32, at 104 (these are the calcium sites 3 and 1) and at 229. The estimated error in co-ordinates is 0.2 A. The deviations between the 2 molecules A and B in the P6, cell and between molecules A and 3 and the R3 molecule are 66 A for C” and about 1 A for all atoms (Table 1). As these exceed the errors, distortions due to lattice packing must. be significant. The relation between the 2 molecules in the asymmetric unit of the P6, cell is an almost exact 2-fold axis. (b) Model

interpretations

Comparison of the model co-ordinates was made by a least-squares superposition of all atoms. Surface accessibilities were calculated with HYBAC (Levit,t, 1974) wit,h a probe radius of 1.4 A and bound water molecules taken into account. B simple dielectric model was used in the calculations of the electrostatic potential of annexin V: the protein was treated as medium wit,h a low dielectric constant, (E = 2) surrounded by the solvent {E = 78). The PoissonBoltzmann equations were solved for this system using the finite-difference method (Klapper et al., 1986; Gilson et

Structure

of Human

Final

re$nement

Annexin

V

685

Table 1

Resolution range (A) Number of independent reflections Completeness (resolution in A) Total number of atoms Solvent atoms R-factor (resolution in d) (8-2.3) (2.58-2.47) (2.38-2.3) Average B-factors (A2) Main-chain Side-chain Solvent Calcium r.m.s. deviation from standard values Bonds (pi) Angles (deg.) Energy (kcal/mol) A. Comparison

and transformations

(i) P6,

--t R3

(MolA)

Matrix:

+ P6,

Matrix:

corresponds

(2.38-2.3)

8.0-2.0 19,927 (8.0-2.0) 2714 202

0.71

0184 0.256 0290 MolA MolA 39 39

0.840 (2.15-2.07)

0.61

(8-2.0) 0.174 (2.25-2.15) 0232 (207-2.0) 0.284 26: MolB 30: MolB

26 29

24 30 33 24

0.014 2.319 - 4249

0.011 1.829 -2894

(iii) P6, (MolB) -0.01559 @02186 - 099964 95922

--+ R3

Matrix:

0.62881 0.77754 -077716 062821 -0.02510 002815 -44011 - 33498 0.9049 (all atoms) 0.6310 (C”)

Vector: r.m.s. deviation:

- 0.006 11 - 003722 099929 -61.075

(MolB)t 0.06303 099801 099768 - 0.06304 -0.02571 0.00035 - 56.752 62.968 1.0485 (all atoms) 0.6295 (P)

Vector: r.m.s. deviation:

t This

0882

0.81633 0.57737 @57758 -0.81604 -@OOOlO - 0.02685 -31.556 44.463 0.9625 (all atoms) 0.6557 (C=)

Vector: r.m.s. deviation: (ii) P63 (MolA)

8.0-2.3 27,422 (&O-23) 5328 277

statistics

to a rotation

of 178.98”

al., 1985; see Tanford & Kirkwood, 1989, for alternative methods).

-000128 -0.02568 -0.99967 153.872

around

an axis with

1957; Karshikov

et al.,

polar

angles 4681”

and 073”

IA:

17-28

IB:

35-42

chain

IC:

47-61

ID:

65-72

IE: IIA:

75-85 88-100

conformation

The four amino acid repeats of annexin V are similarly folded into a compact arrangement of five helixes wound into a right-handed superhelix (for nomenclature, see Huber et al., 1990a). The connections between repeats I and II and III and IV are repeat II-III short, interhelical turns. The connector is an extended segment of about ten residues. Similarly extended is the N terminus up to residue 15. The helices are seven to 15 amino acid residues long. They are rather regular a-helical and were classified by the definition of Kabsch & Sander (1983: i.e. more stringent than that used previously by Huber et aZ. (1990a)). They were quite accurately predicted by Taylor & Geisow (1987) and Barton et aZ. (1991), probably a reflection of their regularity and mostly canonical initiation and termination sequences summarized below:

IIB:

107-l

IIC:

119-133

IID:

137-144

@3 A.

(N-terminal cap by D16 and E17; C-terminal cap by Ca3); (N-terminal cap by D34, E35 and E36; terminated by a hydrogen bond of S44 OG to 40 0 and 41 0; C-terminal cap by R45); (initiated by S46, N-terminal cap by Dll and E278; terminated by a hydrogen bond of T59 OG to 55 0); (N-terminal cap by D64; terminated by a hydrogen bond of S71 OG to 68 0);

3. Results and Discussion (a) Polypeptide

and screw component

16

(break in helix at 90; C-terminal cap by Cal); (N-terminal cap by N106; terminated by a hydrogen bond of SllS OG toll2 0); (initiated by P119, N-terminal cap by El20 and E121); (initiated by S136 OG and a negatively charged cap by

R. Huber

686

et al.

-----

60

AsplSOX Ala293 10 I I I

0

I I I I I I I

0 0

I I I

I I

’

! I

I I I

I I

I I

l I

I

-------,-------j------/

f

x

1

I

I

RI

-------i-------c------

k lo

q

00

I qoal / I $0

/ I I

/

I 0 cp Figwe 1. Ramachandran plot of the R3 molecular structure showing the derived right-handed a-helical conformation around - 60”, -40” in 4 : I). Five residues in left-handed helical conformation that are not glycine residues, A159, K186, W187, D190 and A293, are indicated. Glycine residues are denoted by open squares. -180

IIE:

147-157

IIIA:

169-I 84

11113:

191-200

IIIC:

203-217

IIID:

221-224

IIIE:

232-245

IVA:

247-259

IVB:

2666275

IVC:

281-292

-120

-60

120

El38 and E139; terminated by a hydrogen bond of T145 OG

IVD:

296-303

to

IVE:

306-316

141

0);

helix 157 to 158 ended by Al59 in left-handed helical conformation); (N-terminal cap by D168; 3,0 helical turn at 184 to 186); (N-terminal cap by D190; C-terminal cap by R161); (initiated by S202 and S204, terminated by S217 OG, hydrogen bonded to 213 0); (3,0 helix; X-terminal cap by E222); (N-terminal cap by E234; C-terminal cap by R207); (initiated by P248; C-terminal cap by Ca2); (N-terminal cap by D26.5; terminated by a hydrogen bond of S275 OG to 271 0);

(initiated by 5295 06: hydrogen bonded to S298 OG); (terminated by G317).

(3,,

The Ramachandran plot of Figure 1 documents the predominant) a-helical conformation. The /&structures are associated with turns, the N terminus and the connector between repeat II-III. Five well-defined residues that are not glycine residues occur in an energetically unfavorable lefthanded helical conformation. These residues are: A159, which ends helix IIE and leads to the connector of repeats II and III. Residue 159 is A or mostly G in known annexin sequenees. K186 to W187, which terminate helix IIIA by a half turn of a left-handed helix that allows K186 and W187 to be positioned externally and internally, respectively. D190, which is located at turn IIIA-IIIB. It is invariant except, in t,he second half of annexin VI (~68). A293, which ends helix IVC and initiates t,urn IVC-IVD. Residue 293 is predominantly glycine in annexins.

Structure (b) Family

of Human

relationships

Amino acid sequence homology between members of the annexin family is high from the invariant F15 on, which marks the onset of the globular structure (Table 2). The segment N-terminal to F15 is of variable length. In annexin V it is short and in extended conformation and links repeats I and IV

Sequence

Annexin

V

687

non-covalently. Residue 9 is a conserved I/V residue in all annexins and anchors the N terminus to repeats I and IV, between which it is completely buried. Annexin VII (synexin) is the longest with an extension of 120 amino acid residues with G/Y/P/Q-based sequence motifs (Burns et al., 1989; Doring et al., 1991). The binding properties and

Table 2 alignment of annexins

A32554MSYPGYPPTGYPPFPGYPPAGQESSFPPSG QYPYPSGFPPMGGGAYPQVPSSGYPGAGGY PAPGGYPAPGGYPGAPQPGGAPSYPGVPPG QGFGVPPGGAGFSGYPQPPSQSYGGGPAQV 501016 c29250 SO1786

*

SO0263 A31079

MAKIA DAAGQFFPEAAQVAYQMWELSAV

*

DAAGQFFPEAAQVAYQMWELSAV

*

DAAGQFFPEAAQVAYQMWELSAT

MAKPA MAKPA M

so7434 A27107 A31578 B29250 A29250

MAS MAAS MAAS MAWWKA

G25728 G15397 A32554 so2779 JT0303 A28641 SO2181 A32299

633276 GO4100 G12934

G20407

PLPGGFPGGQMPSQYPGGQPTYPSQPAT MSMVSEFLKQAYFIDNQEQDYVKTVKS MAMVSEFLKQACYIEKQEQEYVQAVKS MAMVSEFLKQAWFIENEEQEYVQTVKS MAMVSEFLKQARFLENQEQEYVQAVKS EFLKQARFLENQEQEYVQAVKS MAVVSEFLKQAWFMENLEQECI MSTVHEILCKLSLEGDHS MSTVHEILCKLSLEGDHS MSTVHEILCKLSLEGDHS IA

10 SO1016MAQVLRGTVTDFPGFDERADAETLRKAMK-i c29250 MALRGTVTDFSGFDGRADAEVLRKAMKG S01786QGAMYRGSVHDFPEFDANQDAEALYTAMKG *SRVELKGTVCAANDFNPDADAKALRKAMKG S00263QGAKYRGSIHDFPGFDPNQDAEALYTAMKG *ARVELKGTVRPANDFNPDADAKALRKAMKG A31079QGAKYRGSIHDFPGFDPNQDAEALYTAMKG *ARVELKGTVRPANDFNPDADAKALRKAMKG SO7434AMATKGGTVKAASGFNAMEDAQTLRKAMKG A27107 AAKGGTVKAASGFNAAEDAQTLRKAMKG A31578 MAAKGGTVKAASGFNAAEDAQTLRKAMKG B29250IWVGHRGTVRDYPDFSPSVDAEAIQKAIRG A29250LWVGPRGTINNYPGFNPSVDAEAIRKAIKG G25728LWVGPRGTINNYPGFNPSVDAEAIRKAIKG G15397WIEQEGVTVKSSSHFNPDPDAETLYKAMKG A32554VTQVTQGTIRPAANFDAIRDAEILRKAMKG S02779SKGGPGSAVSPYPSFDASSDVAALHKAITV JTO303YKGGPGSAVSPYPSFNPSSDVAALHKAIMV A2864lSKGGPGSAVSPYPTFNPSSDVAALHKAIMV S02181YKGGPGSAVSPYPSFNVSSDVAALHKAIMV A32299YKGGPGSAVSPYPSFNVSSDVAALHKAIMV G33276KCTQCVHGVPQQTNFDPSADVVALEKAMTA GO41OOTPPSAYGSVKAYTNFDAERDALNIETAIKT G12934TPPSAYGSVKAYTNFDAERDALNIETAIKT G20407TPPSAYGSVKPYTNFDAERDALNIETAVKT

20

30

R. Huber et al.

688

Table 2 (continued) Ca3

IB

IC

40 S01016LGTDEESILTLLTSRSNAQRQEISAAFKTL C2925OLGTDEDSILNLLTARSNAQRQQIAEEFKTL S01786FGSDKESILELITSRSNKQRQEICQSYKSL *IGTDEATIIDIVTHRSNAQRQQIRQTFKSH SO0263FGSDKEAILDIITSRSNRQRQEVCQSYKSL *LGTDEDTIIDIITHRSNVQRQQIRQTFRSH A31079FGSDKEAILDIITSRSNRQRQEVCQSYKSL *LGTDEDTIIDIITHRSNVQRQQIRQTFKSH SO7434LGTDEDAIISVLAYRNTAQRQEIRTAYKST A27107LGTDEDAIISVLAYRSTAQRQEIRTAYKST A31578LGTDEDAIINVLAYRSTAQRQEIRTAYKTT B29250IGTDEXMLISILTERSNAQRQLIVKEYQAA A29250IGTDEKTLINILTERSNAQRQLIVKQYQEA G25728IGTDEKTLINILTERSNAQRQLIVKHIQEA G15397IGTNEQAIIDVLTKRSNTQRQQIAKSFKAQ A32554FGTDEQAIVDVVANRsNDQRQKIKAAFKTS SO2779KGV0EATIIDILTKRNNAQRQQl-KAAYLQE JTO303KGVDEATIIDILTKRTNAQRQQIKAAYLQE A28641KGVDEATIIDILTKRNNAQRQQIKAAYLQE SO2l8lKGV0EATIIDILTRRTNAQRQQIKAAYLQE A32299KGVDEATIIDILTKRTNAQRPRIKAAYLQE G33276KGVDEATIIDIMTTRTNAQRPRIKAAYHKA GO4100KGVDEVTIVNILTNRSNEQRQDIAFAYQRR G12934KGVDEVTIVNILTNRSNAnRQDIAFAYQRR 620407KGVDEVTIVNILTNRSNVQRQDIAFRYQRR

50

Ca3 * 70 S01016FGRDLLDDLKSELTGKFEKLIVALMKPSRL C29250FGRDLVNDMKSELTGKFEKLIVALMKFSRL S01786YGKDLIEDLKYELTGKFERLIVNLMRPLAY *FGRDLMADLKSEISGDLARLILGLMMPPAH S00263YGKDLIADLKYELTGKFERLIVGLMRPPAY *FGRDLMTDLKSEISGDLARLILGLMMPPAH A31079YGKDLIADLKYELTGKFERLIVGLMRPPAY *FGRDLMTDLKSEISGDLARLILGLMMPPAH S07434IGRDLIDDLKSELSGNFEQVIVGMMTPTVL A27107IGRDLLDDLKSELSGNFEQVILGMMTPTVL A31578IGRDLMDDLKSELSGNFEQVILGMMTPTVL B29250YGKELKDDLKGDLSGHFEHLMVALVTPPAV A29250YEQALKADLKGDLSGHFEHVMVALITAPAV G25728YEQALKADLKGDLSGHFEHVMVALITAPAV 615397FGKDLTETLKSELSGKFERLIVALMYPPYR A32554YGKDLIKDLKSELSGNMEELILALFMPPTY S02779KGKPLDEALKKALTGHLEEVVLALLKTPAQ JT0303TGKPLDETLKKALTGHLEEVVLAMLKTPAQ A28641TGKPLDETLKKALTGHLEEVVLALLKTPAQ S02181NGKPLDEVLRKALTGHLEEVVLAMLKTPAQ A32299NGKPLDEVLRKALTGHLEEVVLAMLKTE'AQ 633276KGRSLEEAMKRVLKSHLEDVVVALLKTPAQ 604100TKKELASALKSALSGHLETVILGLLKTPAQ 612934TKKELASALKSALSGHLETVILGLLKTPAQ 620407TKKELPSALKSALSGHLETVILGLLKTPAQ

IE

ID

IIA

80

Cal

100 SO1016YDAYELKHALKGAGTNEKVLTETIAsRTPE C29250YDAYELKHALKGAGTDEKVLTEIIASRTPE SO1786CDAKEIKDAISGIGTDEKCLIEILASRTNE *YDAKQLKKAMEGAGTDEKTLIEILATRTNA S00263CDAKEIKDAISGIGTDEKCLIEILASRTNE *YDAKQLKKAMEGAGTDEKALIEILATRTNA A3P079CDAKEIKDAISGIGTDEKCLIEILASRTNE *YDAKQLKKAMEGAGTDEKALIEILATRTNA S07434YDVQELQRAMKGAGTDEGCLIEILASRTPE A27107YDVQELRRAMKGAGTDEGCLIEILASRTPE A31578YDVQELRKAMKGAGTDEGCLIEILASRTPE B29250FDAKQLKKSMKGAGTNEDALIEILTTRTSR

60

90

IIB 110

120

Structure

of Human Annexin

V

689

Table 2 (continued) Cal

IIA 100 A29250FDAKQLKKSMRGMGTDEDTLIEILTTRTSR G25728FDAKQLKKSMRGMGTDEDTLIEILTTRTSR G15397YEAKELHDAMKGLGTKEGVIIEILASRTKN A32554YDAWSLRKAMQGAGTQERVLIEILCTRTNQ S02779LDADELRAAMKGLGTDEDTLIEILVSRKNR JT0303FDADELRAAMKGLGTDEDTLIEILTTRSNQ A28641FDADELRAAMKGLGTDEDTLIEILASRTNK S02181FDADELRGAMKGLGTDEDTLIEILTTRSNE A32299FDADELRGAMKGLGTDEDTLIEILTTRSNE G33276FDAEELRACMKGHGTDEDTLIEILASRNNK G04100YDASELKASMKGLGTDEDSLIEIICSRTNQ G12934YDASELKASMKGLGTDEDSLIEIICSRTNQ G20407YDASELKASMKGLGTDEDSLIEIICSRTNQ IIC

IIB 110

Cal *

IID 130

120

140

-

150

S01016ELRAIKQVYEEEYGSSLEDDVVGDTSGYYQ C29250ELRAIKQAYEEEYGSNLEDDVVGDTSGYYQ S01786QMHQLVAAYKDAYERDLESDIIGDTSGHFQ *EIRAINEAYKEDYHKSLEDALSSDTSGHFR S00263QMHQLVAAYKDAYERDLEADIIGDTSGHFQ *EIRAINEAYKEDYHKSLEDALSSDTSGHFR A31079QMHQLVAAYKDAYERDLEADIIGDTSGHFQ *EIRAINEAYKEDYHKSLEDALSSDTSGHFR SO7434EIRRISQTYQQQYGRSLEDDIRSDTSFMFQ A27107EIRRINQTYQLQYGRSLEDDIRSDTSFMFQ A31578EIRRINQTYQLQYGRSLEDDIRSDTSFMFQ B292500MKDISOAYYTVYKKSLGDDISSETSGDFR A29250~MKEIS~AYYTAYKKNLRDDISSETSGDFR G25728QMKEISQAYYTAYKKNLRDDISSETSGDFR G15397QLREIMKAYEEDYGSSLEEDIQADTSGYLE A32554EIREIVRCYQSEFGRDLEKDIRSDTSGHFE S02779EIKEINRVYRDELKRDLAKDITSDTSGDFQ JTO303QIREITRVYREELKRDLAKDITSDTSGDFR A28641EIRDINRVYREELKRDLAKDITSDTSGDFR SO2181QIREINRVYREELKRDLAKDITSDTSGDFR A32299QIREINRVYREELKRDLAKDITSDTSGDFR G33276EIREACRYYKEVLKRDLTQDIISDTSGDFQ G04100ELQEINRVYKEMYKTDLEKDIVSDTSGDFR G12934ELQEINRVYKEMYKTDLEKDIISDTSGDFR G20407ELQEINRVYKEMYKTDLEKDIISDTSGDFR IIE 160 SOlOl6RMLVVLLQANRDPDAGIDEAQ-VE-QDAQA C29250RMLVVLLQANRDPDTAIDDAQ-VE-LDAQA SOl786KMLVVLLQGTRENDDVVSEDL-VQ-QDVQD *RILISLATGNREE/;z;i;,DAQ-VA-AEILE

170

500263

K M L V V L L Q G T R E E'D *RILISLATGHREE/;z;;;,DAQ-VA-AEILE

D V V S'E

D L -VQ-

QDVQD

A31079

K M L V V L L Q G T R E E'D *RILISLATGHREE;;E;;;,DAQ-VA-AEILE

D V V S'E

D L -VQ-

QDVQD

SO7434 R V L V S L S A G G R D E'G A27107RVLVSLSAGGRDEGNYLDDAL-VR-QDAQD A31578RVLVSLSAGGRDESNYLDDAL-MR-QDAQD B29250KALLTLADGRRDESLKVDEHL-AK-QDAQI A29250KALLTLADGGRDESLKVDEHL-AK-KDAQT G25728KALLTLADGGRDESLKVDEHL-AK-KDAQT G15397RILVCLLQGSRDDVSSFVDPA-LALQDAQD A32554RLLVSMCQGNRDENQSINHQM-AQ-EDAQR SO2779KALLSLAKGDRCEDLSVNDDL-AD-SDARA JTO303NALLALAKGDRCEDMSVNQDL-AD-TDARA A28641NALLSLAKGDRSEDFGVNEDL-AD-SDARA SO218lKALLALAKGDRCQDLSVNQDL-AD-TDARA A32299KALLALAKGDRCQDLSVNQDL-AD-TDARA

N Y L D'D

A L -VR

-QDAQD

R. Huber

690

et al

Table 2 (continuedj IIE 170

160 G33276KALVSLAKADRCENPHVNDEL-AE-KDARA G04100KLMVALAKGRRAEDGSVIDYELID-QDARD G12934KLMVALAKGRRAEDGSVIDYELID-QDARD G20407KLMVALAKGRRAEDGSVIDYELID-QDARE IIIA

IIIB

180 190 SO1016LFQAGELKWGTDEEKFITIFGTRSVSHLRK C29250LFQAGELKWGTDEEKFITILGTRSVSHLRR S01786LYEAGELKWGTDEAQFIYILGNRSKQHLRL *IADTPSGDKTSLETRFMTVLCTRSYPHLRR S00263LYEAGELKWGTDEAQFIYILGNRSKQHLRL *IADTPSGDKTSLETRFMTILCTRSYPHLRR A31079LYEAGELKWGTDEAQFIYILGNRSKQHLRL *IADTPSGDKTSLETRFMTILCTRTYPHLRR S07434LYEAGEKKWGTDEVKFLTVLCSRNRNHLLH A27107LYEAGEKKWGTDEVKFLTVLCSRNRNHLLH A31578LYEAGEKKWGTDEVKFLTVLCSRNRNHLLH B29250LYKAGENRWGTDEDKFTEILCLRSFPQLKL A29250LYDAGEKKWGTDEDKFTEILCLRSFPQLKL G25728LYDAGEKKWGTDEDKFTEILCLRSFPQLKL G15397LYAAGEKIRGTDEMKFITILCTRS.ATHLLR A32554LYQAGEGRLGTDESCFNMILATRSFPQLRA S02779LYEAGERRKGTDVNVFITILTTRSYSHLRR JTO~O~LYEAGERRKGTDVNVF'NTILTTRSYPHLRK A28641LYEAGERRKGTDVNVFNTILTTRSYPQLRR S02181LYEAGERRKGTDVNVFTTILTSRSFPHLRR A32299LYEAGERRKGTDVNVFHTILTSRSFPHLRR G33276LYEAGEQKKGTDINVFVTVLTARSYPH-SE 6041OOLYDAGVKRKGTDVPKWISIMTERSVCHLQK 612934LYDAGVKRKGTDVPKWISIMTERSVPHLQK G20407LYDAGVKRKGTDVPKWISIMTERSVCHLQK IIIC

200

IIID

210 220 S01016VFDKYMTISGFQIEETIDRETSGNLEQLLL C29250VFDKYMTISGFQIEETIDRETSGNLENLLL S01786VFDEYL,KTTGKPIEASIRGELSGDFEKLML *VFQEFIKKTNYDIEHVIKKEMSGDVKDAFV S00263VFDEYLKTTGKPIEASIRGELSGDFEKLML *VFQEFIKMTNYDVEHTIKKEMSGDVRDAFV A31079VFDEYLKTTGKPMKASIRGELSGDFEKLML *VFQEFIKMTNYDVEHTIKKEMSGDVRDAFV S07434VFDEYKRISQKDIEQSIKSETSGSFEDALL A27107VFDEYKRISQKDIEQSIKSETSGSFEDALL A31578VFDEYKRIAQKDIEQSIKSETSGSFEDALL B2925OTFDEYRNISQKDIVDSIKGELSGHFEDLLL A29250TFDEYRNISQKDIEDSIKGELSGHFEDLLL G25728TFDEYRNISQKDIEDSIKGELSGHFEDLLL G15397VFEEYEKIANKSIEDSIKSETHGSLEEAML A32554TMEAYSRMANRDLLSSVSREFSGYVESGLK S02779VFQKYTKYSQHDMNKALDLELKGDIENCLT JT0303VFQNYRKYSQHDMNKALDLELKGDIEKCLT A28641VFQKYTKYSKHDMNKVLDLELKGDIEKCLT S02181VFQNYGKYSQHDMNKALDLELKGDIEKCLT A32299VFQNYGKYSQHDMNKALDLELKGDIEKCLT G33276VFQKYTKYSKHDMNKAVDMEMKGDIEKCLT G041OOVFERYKSYSPYDMLESIKKEVKGDLENAFL G12934VFDRYKSYSPYDMLESIRKEVKGDLENAFL G20407VFERYKSYSPYDMLESIKKEVKGDLENAFL IIIE 240 250 S01016AVVKSIRSIPAYLAETLYYAMKGAGTDDHT C2925OAVVKSIRSIPAYLAETLYYAHKGAGTDDHT S01786AVVKCIRSTPEYFAERLFKAMKGLGTRDNT *AIVQSVKNKPLFFADKLYKSMKGAGTDEKT S00263AVVKCIRSTPEYFAERLFKAMKGLGTRDNT

230

Ca2

IVA 260

Structure

of Human

Annexin

V

691

Table 2 (continued) IIIE

Ca2

IVA

240 250 *AIVQSVKNKPLFFADKLYKSMKGAGTDEKT A31079AVVKCIRSTPEYFAERLFKAMKGLGTRDNT *AIVQSVKNKPLFFADKLYKSMKGAGTDEKT SO7434AIVKCMRNKSAYFAEKLYKSMKGLGTDDNT A27107AIVKCMRNKSAYFAERLYKSMKGLGTDDNT A31578AIVKCMRNKSAYFAERLYKSMKGLGTDDDT B2925OAIVNCVRNTPAFLAERLHRALKGIGTDEFT A2925OAVVRCTRNTPAFLAGRLHQALKGAGTDEFT G25728AVVRCTRNTPAFLAGRLHQALKGAGTDEFT G15397TVVKCTQNLHSYFAERLYYAMKGAGTRDGT A32554TILQCALNRPAFFAERLYYAMKGAGTDDST SO2779AIVKCATSTPAFFAEKLHLAMKGAGTRHKA JT0303TIVKCATSTPAFFAEKLYEAMKGAGTRHKT A28641AIVKCATSKPAFFAEKLHQAMKGVGTRHKA S02181TIVKCATSTPAFFAEKLYEAMKGAGTRHKA A32299TIVKCATSHPAFFAEKLYEAMKGAGTRHKA G33276ALVKCATSKPAFFAEKLHMAMKGFGTQHRD G041OONLVQCIQNKPLYFADRLYDSMKGKGTRDKV G12934NLVQCIQNKPLYFADRLYDSMKGKGTRDKV G20407NLVQCIQNKPLYFADRLYDSMKGKGTRDKV

260

IVB 270 280 S01016LIRVMVSRSEIDLFNIRKEFRKNFATSLYS C2925OLIRVIVSRSEIDLFNIRKEFRKNFATSLYS S01786LIRIMVSRSELDMLDIREIFRTKYEKSLYS *LTRVMVSRSEIDLLNIRREFIEKYDKSLHQ S00263LIRIMVSRSELDMLDIREIFRTKYEKSLYS *LTRIMVSRSEIDLLNIRREFIEKYDKSLHQ A31079LIRIMVSRSELDMLDIREIFRTKYEKSLYS *LTRIMVSRSEIDLLNIRREFIEKYDKSLHQ S07434LIRVMVSRAEIDMLDIRAHFKRLYGKSLYS A27107LIRVMVSRAEIDMMDIRANFKRLYGKSLYS A31578LIRVMVSRAEIDMLDIRANFKRLYGKSLYS B2925OLNRIMVSRSEIDLLDIRTEFKKHYGYSLYS A29250LNRIMVSRSEIDLLDIRREFKKHYGCSLYS G25728LNRIMVSRSEIDLLDIRREFKKHYGCSLYS G15397LIRNIVSRSEIDLNLIKCHFKKMYGKTLSS A32554LVRIVVTRSEIDLVQIKQMFAQMYQKTLGT S02779LIRIMVSRSEIDMNDIKVYYQKMYGISLCQ JTO303LIRIMVSRSEIDMNEIKVFYQKKYGIPLCQ A28641LIRIMVSRSEIDMNDIKAFYQKMYGISLCQ S02181LIRIMVSRSEIDMNEIKVFYQKKYGISLCQ A32299LIRIMVSRSEIDMNEIKVFYQKKYGISLCQ G33276LIRIMVSRHEVDMNEIKGYYKKMYGISLCQ GO41OOLIRIMVSRSEVDMLKIRSEFKKKYGKSLYY G12934LIRIMVSRSEVDMLKIRSEFKRKYGKSLYY G20407LIRIMVSRSEVDMLKIRSEFKRKYGKSLYY Ca2 * 300 SO1016MIKGDTSGDYKKALLLLCGEDD* C2925OMIKGDTSGDYKKALLLLCGGEDD* 501786MIKNDTSGEYKKALLKLCGGDD *AIEGDTSGDFMKALLALCGGED* S00263MIKNDTSGEYKKTLLKLSGGDD *AIEGDTSGDFLKALLALCGGED* A31079MIKNDTSGEYKKTLLKLSGGDD *AIEGDTSGDFLKALLALCGGED* S07434FIKGDTSGDYRKVLLVLCGGD,D* A27107FIKGDTSGDYRKVLLILCGGDD" A31578FIKGDTSGDYRKVLLILCGGDD* B2925OAIKSDTSGDYEITLLKICGGDD* A2925OAIQSDTSGDYRTVLLKICGGDD* G25728AIQSDTSGDYRTVLLKICGGDD* 615397MIMEDTSGDYKNALLSLVGSDP* A32554MIAGDTSGDYRRLLLAIVGQ* S02779AILDETKGDYEKILVALCGGQ" JT0303AILDETKGDYEKILVALCGGN*

IVC

IVD 290

IVE 310

320

R. Huber et al.

692

Table 2 (continued) Ca2 * 300 A28641AILDETKGDYEKILVALCGG S02181AILDETKGDYEKILVALCGGM* A32299AILDETKGDYEKILVALCGGN* 633276AIMDELKGGYETILVALCGSDN* G04100YIQQDTKGDYQXALLYLCGGDD* G12934YIQQDTKGDYQKALLYLCGGDD* G20407YIQQDTKGDYQKALLYLCGGDD* Helical SO1016, C29250, S01786, $00263, A31079, S07434, 827107, A31578, B29250, A29250, 625728, 615397, A32554, 502779, JT0303, $28641, S02181, A32299, G33276, G04100, G12934, G20407,

IVE 310

320

regions are delineated including residues that have at least 1 interhelical hydrogen bond. Phospholipid-binding protein: human, Maurer-Fogy et al. (1988). Lipocortin V: rat, Pepinsky et al. (1988). Calcium-binding protein ~68: mouse, Moss ef al. (1988). Calcium-binding protein, ~68: human, Crompton et al. (1988b). Calelectrin: human, Siidhof et al. (1988). Human protein PP4-X: human, Grundmann et al. (1988). Calpactin I heavy-chain: pig, Weber et al. (1987). Endonexin: bovine, Hamman et al. (1988). Lipocortin III: human, Pepinsky et al. (1988). Lipocortin III: rat, Pepinsky et al. (1988). Rat lipocortin-III mRNA, Pepinsky et al. (1988). Human mRNA for vascular anticoagulant-beta (VAC-beta): Hauptmann et al. (1989) Synexin: human, Burns et al. (1989). Lipocortin: guinea pig, Sato et al. (1989). Lipocortin I: rat, Shimizu et al. (1988); Pepinsky et al. (1986). Lipocortin I: human, Varticovski et al. (1988). Lipocortin I: mouse, Sakata et al. (1988). *Lipocortin I: mouse (fragment), Philipps et al. (1989). Pigeon (CoZ. Z&a) calpactin mRNA, H0rsema.n (1989). Bovine calpactin I heavy-chain (~36) protein, mRNA, Kristensen et al. (1986). Human liuocortin II mRNA. Huane et al. (19861. Mouse cafpactin I heavy-chain (p36y mRNk, Saris et al. (1986).

function of the synexin’s N terminus is unknown but may be related to binding to cytoskeletal proteins. The N-terminal segment of 18 additional residues of annexin II (~36) is, however, the welldefined binding site for the small pll protein to form the heterodimer (~36, pll), and probably adopts an a-helical conformation in the oligomer (Johnsson et al., 19886; Becker et al., 1990). About 40 residues in the globular part of the annexin molecules are invariant. Almost all replacements are conservative. There are no long insertions except at the N terminus, as mentioned. One or two-residue insertions occur at the C terminus and in the segment around residue 170 that links repeats II and III. Figure 2 displays the chain tracing as seen along the central local dyad axis (i.e. onto the membrane plane in the membrane-annexin complex) with the invariant residues overlayed. There are 20 buried charged residues, of which 14 are invariant, mostly projecting into the central of the importance of these pore, a reflection elements for maintaining the structure and function of annexins. Residues forming the cores of the four repeats (Table 3) are hydrophobic except for R50 in repeat I. Its charge is balanced by hydrogen bonds to three carbonyl oxygen atoms, L42 0, L84 0 and R45 0. The core residues are highly conserved in all amino acid sequences except W187 in repeat III, which is replaced by a lysine in annexins I and II with

drastic structural consequences discussed below. Table 3 summarizes also the intra-module (interrepeat) contacts formed by the tightly packed repeats II and III and I and IV, respectively. These contacts are predominantly of hydrophobic and neutral polar nature. The intermodule contact generates the central pore discussed iater. Invariant segments are the binding loops for calcium 1 and 2 -G-X-G-T(positions 102 to 105 and 261 to 264, respectively) in repeats II and IV. A homologous loop exists in repeat I in annexin V, where it has calcium (Ca3) bound and in other annexins, but not in annexin I and II (Table 2). Repeat III in annexin V is very differently structured at residues 186 to 189, forming the turn IIIA-IIIB. In this segment, which is homologous to the calcium binding loops in repeats I, II and IV, W187 is turned into the core of repeat III, and the main-chain traces a. rather different course. -4s a consequence, the canonical D and E residues in turn IIID-IIIE of the calcium (-G-X-G-T-, 38 residues, -D/E-) motif move away by more than 15 A. Figure 3 displays the major rearrangement of loops IIIA-IIIB (top, right side) and IIID-IIIE (top, left side). In annexins I and II, W187 is replaced by a lysine residue tha,t must adopt an alternative, probably exposed, position so that the loop is able to bind calcium. These same annexins have a substantial alteration of the calcium motif in repeat 1: which

Structure

of Human

Annex-in V

693

(b)

Figure 2. (a) Stereo view of the C” chain trace with the residues that are invariant in all annexin The view is along the central axis. (b) The view is perpendicular to the central molecular axis.

reads -V/A/T-K-G-V-, 38 residues -A/V and is probably not a calcium-binding site. The presence of three calcium-binding sites seems to be a common feature in annexins, of which two are invariably in repeats II and IV and one may be alternatively in either repeat I (all annexins except I and II) or III (annexin I and III). Annexin VI (~68) is twice as large as annexin V and has eight repeats. In Table 2 the two halves are compared with other annexin sequences. The homology of the two halves is evidence for a structural similarity of each to the four repeat annexins, but the long connector of 18 residues between repeats IV and V does not constrain their relative arrangement. The molecular shape of ~68 seen at very low resolution in the electron microscope (Newman et al., 1989) suggests a lateral association such that all putative calcium sites in repeats I, II, IV, V, VI and VIII (according to the presence of the canonical “calcium” sequence) may be similarly oriented.

sequences overlaid.

(c) Repeat relationships Amino acid sequence alignment of the four repeats shows a few conserved residues, which are summarized here. A (21, 93, 176 and 252) is close to residues 57, 129, 213 and 288; a longer side-chain would collide with these residues and interfere with proper packing of helices A and C. L (24, 96, 179 and 255) is a core residue in all repeats. A (27, 99, 182 and 258) is too close to residues 34, 106, 190 and 265 to allow a longer side-chain; it would interfere with proper packing of helices A and B and the formation of the calcium loops in repeat I, II and IV. G-T (32-33, 104-105, 188-189 and 263-264) are calcium sites in repeats I, II and IV, but not in repeat III. R (45, 117, 201 and 276) forms a buried salt link to D (20, 92, 175 and 280). The three former aspartate residues are homologous, but D251 is replaced by L in repeat IV; its role is taken by D280.

R. Huber

694

3. Superposition

Figure

et al.

of C” chain tracings of repeats I to IV. Residues 3 to 87 (I), 88 to 167 (II), 168 to 246 (III) Dl; -, D2; ------, D3; ......, D4.

and

247 to 318 (IV) are drawn. -,

(d) Buried

Figure 3 is an overlay of the Cc-chain tracing of the four repeats of annexin V. The helical segments and their spatial arrangement are closely similar, except for the shorter helix IIID due to the rearrangement of loop IIID-IIIE mentioned above. Repeat II is connected with III by a C-terminal extension, which may also have been drawn as part of repeat III; suggesting homology to the N terminus of repeat I.

charged

residues

Twenty-one Asp, Glu, Arg and Lys residues have an accessibility close to zero. They are summarized in Table 6, together with their side-chain interactions. Many form internal salt bridges, More than half of them are conserved. Residues forming part of the central channel are marked.

Table 3 Conserved

core and contact

residues

(i) Core

residues

Repeat A21

I L24

A27

MM28

138

L41

L42

R50

I53

B-57

L65

L69

k73

I81

iv!&

Repea.t A93

II L96

A99

LlOO

LllO

I113

1114

L122

1125

Y129

L137

V141

T145

L153

L157

Repeat Al76

III L179

AlS2

(~1~7)

F194

I197

F198

L206

V209

(~~1~)

Y213

I221

1225

L237

v241

Repeat IV A252 L255

A258

M259

L269

V272

M273

L281

I284

F288

L296

1300

L312

C316

when

bracketed)

(Residues

in columns

(ii) Inter-repeat Repeat Till Repeat El91

contacts

II interface At15 Slt6 III interface I195 G199

(i) Inter-repeat Repeat L5

are

contacts

Repeat IV interface D266 L269 1270

Module A83 (Underlined

II

the 4 repeats

alzd

except

between

repeats

Y148

Y149

Ml52

L153

V155

L1.56

R161

Dl62

Dl64

T200

S202

N232

L233

L236

A239

V240

s-243

1244

repeats

I and

between

I interface R6 T8

(ii) Inter-m.o&le Module II, III S88 Y91

homologous between

(T224)

1244

I11

T24?

IV

V9

E36

L39

T40

T43

S46

R50

K76

F77

LB0

A83

L84

V274

S277

E278

F282

N283

D307

Y308

K310

A311

L3P4

5315

6317

E95

m

V109

m

1113

Sll6

m

T118

R217

A239

K242

S243

Y250

L251

Y257

m

H267

7’268

R271

V272

V274

5275

contacts

interface J&2 Y94

I, I‘\i interface K86 P87 1247 residues

indicate

charged

E263

residues

T254 within

the central

channel)

m

1279

1)2RO

Structure (e) Internal

of Human

solvent molecules

Forty-eight of the bound water molecules in the R3 crystal form are inaccessible. They are shown in Table 4, together with their co-ordination with other groups. Almost all of them were recognized in early refinement steps by their well-defined electron densities. Buried water molecules are located predominantly in the central pore and are indicated. Most of them are conserved and occur also in either or both molecules of the P6, form. They trace out a path through the molecule that marks sites of loose polar protein-protein packing. They may delineate the ion conduction pathway but may not coincide exactly with intermediate ion positions, should

Annexin

V

695

these exist, as their co-ordination and charge requirements are quite different.

(f) The calcium

sites

Annexin I binds about four Ca2+ with dissociation constants in the micromolar range (Schlaepfer & Haigler, 1987; Ando et al., 1989). In the presence of phospholipid the affinity for calcium is strongly increased (Powell & Glenney, 1987). Calcium binding under conditions of high salt concentration is expected to be much weaker. By using very high calcium concentrations, however, we were able to localize the binding sites in the crystals.

Table 4 Buried Name 404 405 406 408 409 410 411 414 415 416 417 418 419 420 428 431 432 433 436 438 439 440 444 460 462 466 467 468 469 477 486 488 489 511 513 631 638 639 643 650 653 655 667 670 678 702 713 715

Hydrogen

bonded

counter

water molecules

neighbors

Tlll OGl, N232 0, N232 ND2, 620 Q235 NE2, Q235 0, 458, 406 Y250 OEH, 486, 405 N160 N, V155 0, 409 G199 0, 408, 678 Rl61 NEH2, All5 0, 511, 672, 715 R245 NEH2, F210 0, 1221 N, Q220 OEl R117 NEHl, R117 NEH2, El12 OEl Al82 0, T189 0, T189 N, T189 C A93 N, R89 0, 417 S88 0, D92 N, 416, 639 468, 517 D92 ODl, R276 NEHl, S275 0 R276 NEH2, El21 OE2, S116 0, 444 R25 NEHl, D68 ODl, 477 N47 ODl, 501, 642, 649 A48 N, E278 OE2, 472, 649 S54 0, L65 N T74 N, 435, 711 Y91 OEH, E95 OE2 K86 0, A83 0, 440, 468 R50 NEHl, 439, 469 420, 445, 653, 673 K76 NZ, D266 ODl, T264 OGl, 461 S275 0, E278N, 469, 516 544 0, TlO 0, 422, 663 5277 OG, E278 OEl, R50 NEHl, R50 NEHP 418, 439, 469 440, 462, 468 E72 OEl, M28 0, 550 02, 428 K108 0, 406, 487, 488 El12 OE2, 486 E95 OEl, E95 0, 480 1114 0, R117 0, 410, 678, 715 Y213 OEH, F180 0, El84 N D92 OD2 A249 0, N291 ODl, 656 D92 ODl, 417, 601, 712 R285 0, L296 N, 551, 655 R285 NE, 551 03 D280 ODl, 444, 494 E318 0, 551 04, 643 Q51 OEl, M85 0; 510, 708 R271 NEHl, R271 NEHS, T268 OGl L156 0, 409, 511, 715 T8 0, L315 0, 495, 723 L90 0, Y133 OEH, 509 Rl61 NE, 410, 511, 678

in R3 Channel

Homologs

in P63

X

6588,

648s

X X X X

615A, 8528, 6228 704B

629B 725B

X

858B

736B 608A, 607A 801A

627B

611A

6578, 609A

650B

8378, 776B 734B

742B

X X

630B

X

800A

X

851A,

813B

R. Huber

696

Figure 4. Superposition of the calcium phospholipase A,; e, annexin.

binding

et al.

site of phospholipase

Both crystal forms grew in the presence of about 5 mM-calcium in ammonium sulfate at pH 8%. They were harvested into 3lvr-ammonium sulfate (pH 85), 1 mM-CaClz. The concentration of free calcium is probably lower in the presence of ammonium sulfate. The hexagonal crystals cracked when transferred into solutions with a high calcium concentration. In this crystal form the calcium-binding sites 1 and 3 of molecule A and 2 and 3 of molecule B participate in tight lattice contacts, calcium sites 1A and 3B are only 6.9 A apart. The calcium ions and their co-ordinating peptide segments are not well defined in the hexagonal crystals. The rhombohedral crystal form, however, tolerates high calcium concentrations. The crystals develop cracks initially when transferred into solutions with high calcium concentration but anneal, whereby the c-axis shrinks (Huber et ai., 1990b). We find calcium bound with high occupation (Q75) at sites 1 to 3 and wit>h low occupation (O-5) at sites 4 and 5. All have low temperature factors.

A, with

site Cal

of annexin

Calcium prefers hept,a-co-ordination by oxygen ligands located approxima,tely at the vertices of a pentagonal bipyramid (Strynadka & James, 1989). This ligand geometry is observed for Cal and Ca3. Gal is hepta-co-ordinated to the three earbonyl oxygen atoms of LlOO 0, G102 0 and G104 0, the bidentate carboxylate group of Dl44, and two wat’er molecules 481 and 478. The apices of the bipyramid are LlOO 0 and water 478. Ca2 and Ca3 are similarly bound: Ca2 to M259 0, G261 Q and G263 0, the bidentate D303 carboxylate group and water 483 (a 2nd water molecule is missing but there is weak density at the expected position); Ca3 to M28 0, G30 0 and G32 0, the bidentate E72 and two water molecules 403 and 706. The latter has weak density. It is close to sulfate 550, which may mark the phosphoryl-binding site. These t’hree sites had been shown in Figure 4 of Huber et al. (1990b), but the co-ordinating water molecules were defined completely only in subsequent refinement. The average calcium-oxygen distance is 2.42 A.

Table 5 Buried

Name D20 R45 R50 D68 E72 E78 D92 E95 El12 RI17 El21 D140 D144 D175 E253 D265 R271

R276 D280

R285

Hydrogen

charged

bonded

residues,

V. ----.

side-chain

interactions

neighbors

ODl-(R45NE; R45NEH2), OD2-(R45NEH2, 425, 426) NE-(D200Dl), NEHl-(S440G), NEHS-(D200D1, DZOODZ) NE-(L420), NEHI-(LH840, 444, 467), NEHS(R450, 467) ODZ-(R63NE, 428), OD2-(625) Calcium 3 site Calcium 5 site ODI-(RllWE, 419, 639), ODZ-(R117NEB2, 631) OEl-(R271NEH1, H267NE2, 489), 032-(438) OEI-(R27lNE, 414), OE2-(R271NEH2, 488) NE-(DgZODl), NEHI-(SllBOG, 414), SEH2-(D920D2, 414) OEl-(601), OE2-(TllSN, T1180G1, 420) ODI-(Yl29OH, S135OG) ODl-(T1050Gl). OD2-(481, 482) OEl-(H205NE2, RSOlNE), OE2-(R201NEHZ) OEl-(K24ZNZ), 03%(656, 722) ODl-(T268N, 613), OD%(T2680Gl, 443) ?V’E:-(E1120El), NEHI-(E950E1, 670), ?JEH2-(E1120E2, TZ680G1, 670) NE-(D2800D2), NEHl-(419, 420), NEH2-(D28OOD1, 420) ODl-(R276NEH2, 653), OD2-(R276NE) NE-(804551, 650), NEWI-(C3160, E3180El), NEH2-(S04551, E3180E2)

Channel residues

x X X X

Conserved residues

f

X

X X X X

f

Structure

of Human Annexin

A model of the annexin phospholipid interactions is proposed below, based on the structural relation between the calcium site of phospholipase A, (Verheij et al., 1980) and Cal to Ca3. A superposition of the phospholipase A, calcium loop with the Cal site as representative of all three calcium sites in annexin V is shown in Figure 4, demonstrating the remarkable similarity of this segment. In phospholipase A, the calcium is sequestered by a loop with sequence -X-X-G(30)-X-G(32)and aspartic acid residue 49 providing three carbonyl oxygen atoms and two carboxylate proteinaceous oxygen ligands (Verheij et al, 1980). The two remaining co-ordination sites are occupied by phosphoryl- and ester-oxygen atoms respectively, in a substrate analog inhibitor complex (Thunnissen et al., 1990; Scott et al., 1990). The loop and the aspartic acid residue match in structure (and calcium co-ordination with sequence!) the -K-G-X-G-T- (38 residues) -E/D-, of Cal and similarly Ca2 and Ca3 in annexin V. Otherwise phospholipase A, and annexin V are unrelated. Also the general location of the conserved calcium loop is different, very exposed in annexin and in a depression in phospholipase A,. Basic residues, lysine residues 101, 260 and 29, are close to the Cal to Ca3 sites, respectively. These lysine residues are either invariant in all annexins (K260) or strongly conserved, for example KlOl (except in annexin VI (~68)) and K29 (except in annexins I and II, which probably do not have calcium in repeat I). In addition there are residues K97, Y256, R25 and R63 close to the putative phosphoryl-binding sites at Cal to Ca3, respectively, making this site even more basic. Calcium sites 4 and 5 in the rhombohedral form were discovered by their high affinity for lanthanum (difference electron densities 260 for lanthanum at Ca4 and 17a at Ca5, respectively; see Huber et al., 19906) but have also calcium bound with low occupation. These sites are more open and possessonly three proteinaceous oxygen ligands: T33 0 and the bidentate carboxylate of E35 (Ca4) and K70 0, L73 0 and the (monodentate) carboxylate of E78 (Ca5). Co-ordinating water molecules (498, 499 and 500 for Ca4, 605 for Ca5) are also observed. E35 and E78 are conserved except in one or the other half of annexin VI (~68). The Ca4 site is very acidic owing to the presence of spatially close D307, D34 and E36, which are conserved in annexins. Ca4 and Ca5 might also be phospholipid binding sites. The observations that they are strong lanthanumbinding sites and that rare earth ions at low concentration block channel activity (R.B., unpublished results) argue for a role in ion transport. Other lanthanum-binding sites with lower occupancy are at Cal (60) and Ca2 (70) (Huber et al., 1990b) and at G188 0 and the (bidentate) El91 carboxylate (40) and at D226 0 and its carboxylate group (70), which is, however, not well ordered. All calcium and lanthanum-binding sites are at the convex face of the molecule. They are shown in Figure 5 superimposed on the C” chain.

V

697 (g) Phosphorylation sites

Annexin I and II (lipocortin I and II) are substrates of the epidermal growth factor (EGFT) kinase, the kinase encoded by Rous sarcoma virus oncogene (p60VJrc)and protein kinase C. The sites of phosphorylation are located in the N-terminal segment at Y6 and X8 (annexin V enumeration of Table 2) (Gerke & Weber, 1984; Glenney & Tack, 1985; Schlaepfer & Haigler, 1987; Johnsson et al., 1988a). Effects of phosphorylation on calcium and phospholipid binding are small and different for different. annexins and phosphorylations (Powell & Glenney, 1987; Ando et al., 1989). Other properties such as ion conduction have not been measured. In annexin V, residues 6 and 8 are located in the extended N-terminal segment. T8 is largely buried (10% accessibility) between repeats I and IV, close to 315 0 and 316 0 to R285, which could compensate the negative charge of a phosphorylated serine. The phosphoryl group could be accommodated without substantial structural rearrangement. R6 is on the surface (40% accessibility) in stacking interaction with F282, which could be maintained by Y6. A phosphoryl group would be solvent exposed. The N-terminal segment is close to the (exit of the) central pore and, although not directly blocking it, may alter calcium flow when phosphorylated. (h) Membrane annexin

interaction

The comparison of the high resolution crystal structure of annexin V with low resolution electron diffraction image analysis of phospholipid layer bound annexin V (Brisson et al., 1991) provides evidence that there are no major structural rearrangements between these two forms. Surface loop or side-chain motions, however, would not have been seen in the low resolution electron micrographs. To localize phospholipid binding sites in the crystals, we soaked the crystals with short-chain diacylphosphatidic acids, but were unsuccessful in detecting them in the difference Fourier map. The pronounced structural similarity of the annexin calcium loops 1 to 3 with the calcium phospholipid binding site of pancreatic phospholipase A,, however, suggestssimilar binding by replacement of the water molecules in annexin V by the phosphoryl and ester-oxygen atoms. As mentioned above, there is a conserved basic site close to the suggested phosphoryl-binding site, a possible determinant for acidic phospholipid binding (Reutelingsperger et al., 1988; Tait et al., 1989). This interaction may also explain the strong co-operativity between calcium and phospholipid binding, implying direct protein phospholipid interaction in addition to the Ca2+-mediated binding. Assuming planar dimensions of 65 A2 for a phospholipid molecule (Miihwald, 1990), annexin V covers an area of about t Abbreviations used: EGF, epidermal r.m.s., root-mean-square.

growth

factor;

698

R. Huber

et al.

(a)

Figure 5. (a) The calcium 1 to 5 binding sites and the calcium ligands the central dyad axis. (b) Seen onto the central axis.

26 phospholipids. Annexin requires acidic phospholipids for membrane attachment. An estimate of two to five molecules (Schlaepfer et al., 1987) is compatible with binding to three sites, Cal to Ca3. There are no structural data available for the membrane part in the annexin V membrane complex, but phospholipid monolayers show an increase in surface pressure upon incubation with annexin VI (Newman et al., 1989) and annexin V (R. Berendes, A. Burger & D. Voges, unpublished results). The generation of gated ion channels by annexin VII and V (Burns et al., 1989; Rojas et al., 1990; R.B., unpublished results) is also evidence for a profound membrane rearrangement. We proposed that the proximal and possibly also the distal membrane leaflet may be disordered, leading to lateral expansion and (non-selective) ion permeability, and suggested that the ion channel and gating properties of annexin V reside in the protein. Disorder may be caused by specific calcium

superimposed

on the

c” chain

trace

seen along

phospholipid interactions in a geometry dictated by the protein structure and by the electrostatic potential of the protein. It is known that external electric fields can invoke membrane pore formation (electroporation) under certain conditions (Neumann, 1988). The electrostatic potential calculations on annexin V (Marshikov et al., 1991) show that the protein exerts a strong electric field in the region of the protein-membrane interface, and could thus contribute to membrane rearrangement through the electroporation effect. In a wider context the annexin membrane interaction may be compared with other systems binding to, or being activated by, micellar or membraneous fphosphojlipids, such as phospholipase A, and lipases. Phospholipase A, and t,he resemblance of its calcium-binding motif to the annexin motif have been discussed. In the complexes of pancreatic phospholipase A, with monomeric substrate analog inhibitors binding sites for the acyl side-chains have

Xtructure

of Human

been defined. Binding occurs in the absence of a protein main-chain rearrangement (Thunnisen et al., 1990; Scott et al., 1990). With polymeric substrates phospholipid is believed to be partially drawn out of the membrane surface towards the enzyme’s active site (for a review, see Blow, 1991). In the annexin V structure there is no obvious hydrophobic binding site for the bis-acyl side-chains of the phospholipid, in accord with the failure to detect diacyl phosphatidic acids binding; this suggests that they may remain associated with the membrane’s lipid portion. In triacyl glycerol lipases the active site is buried, but becomes accessible after a major movement of a helical “lid” that occurs when a monomeric substrate analog inhibitor is bound. A similar rearrangement is believed to occur at the oil-water interface whereby the substrate is partially drawn out of the oily surface (Brzozowski et al., 1991; Winkler et al., 1990; for a review, see Blow, 1991). Features of these systems that are in common with annexins are the peripheral polymer protein interactions focussed in few specific binding sites. Protein cell-surface association related to annexins seems to exist with Escherichia coli enter0 (cholera)-toxin whose structure in crystalline form has recently been analyzed (Sixma et al., 1991). A pentameric B5 substructure binds to gangliosides at the cell membrane surface with the central pentahelical channel normal to it (Mosser $ Brisson, 1991). It is structurally similar to the pentameric channel of the capsid in riboflavin synthase (Ladenstein et al.; 1988). The ganglioside binding sites and the binding site for subunit A are on opposite faces of the B pentamer (see Fig. 3 of Sixma et al., 1991).

(i) Channel formation Local ion permeability and formation of tight seals at the annexin membrane interface are complementary requirements for channel formation. If, as we suggest, calcium phosphoryl group interaction and the protein’s electrostatic field lead to local disorder, the membrane area associated with Cal to Ca3 of annexin V may be ion permeable. It encompasses Ca4 and Ca5, which are strong lanthanum and weak calcium-binding sites. Lanthanum blocks channel activity in micromolar (R.B., unpublished results), concentrations suggesting that these sites are intermediate binding sites for calcium ion conduction. Direct experimental evidence for the ion conduction pathway is not available. It may be contained in a single molecule or in an oligomer. The trimers in the R3 crystal form and in the annexin phospholipid layer complex are indeed conserved and suggest a preferred mode of lateral association (Brisson et al., 1991). However, observations suggest that the central molecular axis is the ion conduction pore: the invariant calcium-binding sites 1 and 2,

Annexin

V

699

symmetrically arranged around the molecular axis mediate an intimate contact with the membrane; almost all residues forming the central pore (Fig. 2), which is highly hydrated, are conserved (Fig. 6); half of all buried charged residues participate in formation of the pore (Table 5); similarly more than half of all buried water molecules are localized in the pore and were found conserved in both crystal forms (Table 4). We propose therefore that the chain of water molecules from Ca5 on through the central pore marks the ion conduction pathway. Figure 6 is a view of it including bound water molecules. From water 438 on (4th from top) almost all of them are buried and conserved in the different crystal forms (Table 4). They indicate loose protein-protein packing and permeability of the central pore, a necessary but insufficient condition for ion conduction. Even neglecting bound water molecules, free diffusion through the pore is not possible and ion permeation would require some side-chain rearrangements. Arg271, Glul12, Glu92, Argll7, Asp92, Arg276, Glul21 and Asp280 seem to be particular impediments. Residues forming saltbridges across the pore are Arg271-Glu112, Argll7Asp92 and Arg276-Asp280 and may constitute the gate(s). Figure 6(c) illustrates this. The gate(s) may open by alternative salt-bridge formation (Karshikov et d., 1991). The Asp and Glu residues (6 out of 9 buried charged residues) in the pore may play an important role in stabilizing cations on transit (Table 5). Three-dimensional structures of other biological ion channels have not been determined, but a wealth of amino acid sequences, mutational and functional data are available (Numa, 1989; for a review, see Gordon, 1990). The pore of the potassium channel is thought to be associated with an extended rather than helical segment of hydrophobic nature and arranged as an eight-stranded b-barrel within the tetrameric aggregate (for reviews, see Miller, 1991; Stevens, 1991). This suggestion is based on mutational data (Yool & Schwarz, 1991; Hartmann et al., 1991; Yellen et al., 1991). Sodium and calcium channels have a similar structure according to sequence homology (Numa, 1989). The annexin pore is structurally very different from the putative potassium channel pore, but similarly highly charged peptides derived from the nicotinic acetylcholine receptor sequence have been found to form discrete conductance ion channels (Gosh & Stroud, 1991).

(j) Voltage dependent gating A change in the lifetime of open and closed states is the basis of transmembrane voltage-dependent gating and implies the presence of a voltage sensor, i.e. charges or equivalent dipoles, which may move in the electric field (for a review, see Hille, 1984). In annexin V the open state is favored with increasing negative (and less so positive) intracellular holding potential.

700

R. Huber et a!.

ib)

Structure

of Human

Annexin V is strongly electrically dipolar with the positive and negative poles located inside the modules (II and III) and (I and IV), respectively (Fig. 1 in Huber et aE., 1990b). An electric field may alter their relative orientations, thereby changing the diameter of the central cavity. Similar tilting motions have been envisioned for the penta-helical pore of the nicotinic acetyl choline receptor channel (Galzi et al., 1991). An electric field will also act on the charged residues in the pore: the electrostatic energy corresponding to a transmembrane potential of 60 mV (an ion current is observed at this and lower voltages) is about 1.3 kcal/mol (1 cal = 4184 J) and insufficient to break salt linkages; it may, however, favor alternative arrangements of ion pairs corresponding to open or closed ion conductance states (see the gate model of Pig. 6(c)). Voltage-gated potassium channels occur in three states: closed, open and inactivated. The latter is thought to be generated by a physical blockade of the ionic pore by the N terminus of the protein, which was shown to be directly involved in this process (Zagotta et al., 1990; for a review see Miller, 1991). The close association of the N terminus of annexin V with the central pore is suggestive of a similar role, particularly so in annexin II, where the N terminus is the binding site for the small pll protein (Johnsson et al., 19886; Becker et al., 1990).

Annexin

The financial gemeinschaft

activity

Annexins were not known to possess enzymatic activity apart from their property of voltage-regulated channel conductance discussed above. inositol- 1,2-cyclic phosphate Recently, however, (ICP) 2-phosphohydrolase enzymatic activity of annexin III (lipocortin III) was described (Ross et al., 1990). The activity is stimulated by phospholipid binding and requires divalent metals, manganese or magnesium ions. An active site has not been defined but may be associated with the annexin’s metal (i.e. calcium) binding sites. In the presence of phospholipid the Cal to Ca3 sites are likely to be occupied by calcium and phospholipid and unavailable for other metal and ICP binding. The lanthanum sites (Ca4 and Ca5), however, might have affinity for Mn” and Mg2+ ions and could indicate the active sites. In annexin III amino acid sequences a histidine residue 79 is adjacent to E78, which co-ordinates Ca5 and lanthanum (Table 2). H79 may play a role in catalysis by analogy to the key roles of histidine in all known phosphohydrolases.

701

support of the Deutsche ForschungsHul69/11-1 and Me976/2-1 is gratefully The co-ordinates have been deposited with

acknowledged.

the Brookhaven Data Bank. public by December 1992.

They

will

be available

to the

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Figure 6. (a) Stereo-view of the central pore including the charged residues The water chain is drawn from Ca5 on. The view is along the pore axis. (b) water molecules are: from top, 605, (Ca5), 434, 437, 619, 438, 631, 489, (c) Scheme of the proposed channel and its gates. The electrophysiological a strong least 2 open (zol, zo2) and 2 closed (zcl, zc2) life times that exhibit times implies 2 conformational transitions leading to opening/closing of the R. Berendes, A. open (0) and 2 different closed states (cl, c2) (A. Karshikov, unpublished results). In this model we associate the gates 12 and 34 in C,

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Annexin

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by A. R. Fersht