Biochimica et Biophysics Acta 960 (1988) 245-252

Elsevier

245

BBA 52822

Conformational analysis of lipoxin A, lipoxin B and their trans-isomers

Robert Brasseur a, Michel Deleers b, Jean-Marie Ruysschaert a, Bengt Samuelsson ’ and Charles N. Serhan d

a Laboratoire de Chimie Physique des Macromolecules aux Interfaces, Universite Libre a’e Bruxelles,

b Centre de Recherches, Secteur Pharmaceutique, Braine-Z’Alleud (Belgium) ’ Department of Physiological Chemistry,

Karolinska Znstitutet, Stockholm (Sweden) and d Hematologv Division, Department of Medicine,

Brigham and Women’s Hospital and Harvard Medical School, Boston, MA (U.S.A.)

(Received 12 June 1987)

Key words: Lipoxin; Conformational analysis; Theoretical model; Lipid-ion interaction; Arachidonate metabolism

Lipoxin A and lipoxin B (LXA and LXB) are fonned from the oxygenation of arachidonic acid by interactions between the S- and 15lipoxygenases of human leukocytes. Each compound displays highly stereospecific biological actions. Here, we present a computational description of the following compound% lipoxin A, (5S,6R,15S)-trihydroxy-7,9,13-trans-ll-cis-eicosatetraenoic acid; 11-trans-lipoxin A, (5S,6R,15S)-trihydroxy-7,9,11,13-trans-eicosatetraenoic acid; lipoxin B, (5S,14R,15S)-trihydroxy- 6,10,12-trans-8-&r-eicosatetraenoic acid; and &trans-lipoxin B, (5S,14R,15S)-trihydroxy-6,8,10,12-trans- eicosatetraenoic acid. The analyses considered van der Waals energy, electrostatic interactions, torsional potential, and alterations in electrostatic forces. Additional analyses were carried out with each of the four compounds forming complexes with one calcium ion. Each compound gave very different conformers. Both lipoxin A and lipoxin B can form globular conformations, while their all-trans isomers form rigid extended structures. When complexes with each of these compouuds and one calcium ion were examined (i.e., (LXA),Caz (11-tmnr-LXA),Ca), both LXA and LXB formed several flexible conformations including crumpled, wrapped or extended conformations. In this situation, LXA showed a higher probability than LXB to wrap afound one Ca ‘+ In contrast, the two all-trans isomers always lead to extended conformations. . Results from the present study illustrate that changes in the stereochemistry of LXA and LXB lead to unique conformations which may underlie the different biological actions of these compounds. Moreover, they indicate that the conformations of eicosanoids can change while in aqueous or hydrophobic environments (i.e., biomembranes).

Introduction

Oxygenated derivatives of arachidonic acid dis- play a diverse array of biological activities which

Abbreviations: 15-HETE, 15-hydroxy-5,8,11,13-eicosatetra- enoic acid; LXA and LXB, lipoxin A and lipoxin B, respec-

tively.

Correspondence: C.N. Serhan, Hematology Division, Depart- ment of Medicine, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115 U.S.A.

are of importance in both normal and pathophysi- ological circumstances [ 1,2]. Recently, a new series of lipoxygenase-derived products were isolated from activated leukocytes and termed lipoxins (lipoxygenase interaction products) [3-51. These compounds arise by interactions between the 5- and 15-lipoxygenase pathways, and contain a con- jugated tetraene as a distinguishing feature [4]. One biosynthetic route involved in the generation of LXA and LXB has been identified, and the complete stereochemistry of these compounds and

OOOS-2760/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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6S-ll-trans-LXA KS-8-tram-LXB

OH II-trans-LXA &tnns-1x8

&I Llptxin A Lipoxin B

BS-LXA

Fig. 1. Scheme of formation of lipoxins by human leukocytes. The stereochemistry of lipoxin A, lipoxin B. and their isomers shown

here has been established [5,6]. The double bond geometry of the 5(h)-epoxide tetraene intermediate has been proposed.

their isomers has been determined [5,6]. Upon activation, human leukocytes can utilize exoge-

nous 15-HETE to enzymatically generate (5S)-hy- droperoxy-(15S)-hydroxy-6,13-trans-8,1l,cis-eico- satetraenoic acid, which can undergo enzymatic dehydration to form a 5(6)-epoxide tetraene inter-

mediate. This intermediate may give rise to either lipoxin A ((W,6R.15S)-trihydroxy-7,9,13-truns- 11 -c&eicosatetraenoic acid} or lipoxin B (( 53,

14R,15S)-trihydroxy-6,10,12-trans-8-cis-eicosa~et- traenoic acid) by different mechanisms (Fig. 1). Both LXA and LXB possess discrete biological actions which are highly stereospecific and unlike those evoked by other eicosanoids [&lo]. For example, LXA stimulates granulocyte migration at nanomolar concentrations without provoking ag- gregation [lo]. LXA also displays spasmogenic activities and, at submicromolar concentrations, elicits long-lasting contractions of lung strips. This compound also provokes vasodilation in both the hamster cheek pouch [ll] and in the renal micro- circulation of the rat [12]. In the rat, LXA aiso induces glomerular hyperperfusion, hypertension and hyperfiltration [12]. LXA activates isolated protein kinase C in vitro 191, and both LXA and

LXB block human natural killer cell cytotoxicity [7,8]. In each biological system, the all-trans iso- mers of LXA and LXB proved to be less active

than either of their cis double-bond-containing counterparts.

Conformational analyses have been extremely useful in conceptualizing the basis for molecular

OH 0

OH

OH OH

OOH

OH

Fig. 2. Initial conformations of LXB. 8-trans-LXB, and num-

bering of Ca2+ complexes taken as examples for the four

molecules.

241

recognition, interactions between molecules, and probing the structure-function relationships in biological systems [13]. Here, we present confor- mational analyses of lipoxin A, lipoxin B, and their all-truns isomers to gain an appreciation of the different conformations that eicosanoids can maintain in various biological environments.

Materials and Methods

The theoretical conformational analysis (molec- ular structure analysis) of each compound and their possible Ca complexes is based on a semi- empirical procedure described in Refs. 14-16. In this approach, the total conformational energy is

Lipoxm A 11-trons-LXA

lnltiol structure Inltlal str¶Jcture

Lipoxin I3

Inhal structure Initial structure

1. 2. 3. 4, 5 1. 2. 3. 4. 5

I 99%

5. 6. 8. 9 /gw144qo 53%

” 7”

42’ \,4

i \ /I\ 6801. 22% I y i’ /% 7l

Product of probatilitPs

36% 12% 3% 31% 4% 3%

* f I C

34% 23% 21%

I I Product of probabilities

I 30% 20% 1’8%

t

I

Scheme I. Structure trees of each lipoxin after successive conformational analyses bearing on indicated rotational angles with the

indication of the degeneration after minimiza tlon of energy function into an extended (I) or a cryptic (C) form.

248

calculated as the sum of all the contributions due to the van der Waals interactions, torsional poten-

tials and electrostatic interactions [17]. The con- formational energy is calculated for a large num-

ber of conformations in a systematic analysis structure tree for each compound [18-201, or on

one-half of the possible Ca complexes leading to a higher symmetric structure. The conformers which

yield the highest probability were then submitted to the energy function minimization procedure [21] with a precision of 10” on each torsional angle. The values used for the valence angles, boundary lengths, atomic charges, torsional poten-

tials and energy values for the van der Waals interactions are those currently used in conforma-

tion analysis [16,22-241. The systematic and mini-

Fig. 3. Stereoscopic views of the most probable conformers of LXA (a), 11-trans-LXA (b), LXB (c) and 8-trans-LXB (d)

249

Fig. 3 (continued).

mization procedures were made in a medium of intermediate dielectric constant representative of the membrane/water interface [25-261. When complexes with Ca2+ were formed, the transfer energies of each part of the molecule [23] allowed us to calculate the hydrophilic and hydrophobic gravity centers, and to orient the molecular com- plex [17,18,26].

Calculations were performed on an Olivetti M24 computer equipped with the 8087 arithmetic coprocessor using the PC-MSA@ program (Molec- ular Structure Analysis). The computer is coupled to a plotter using the PC-MGM@ program (Molecular Graphic Manipulation). The internal energy of each conformer is calculated in approx. 2 s.

250

Results and Discussion

Upon activation, human leukocytes transform exogenous 15HETE to lipoxins, an event which may occur during cell-cell interactions at in-

flammatory loci [6]. Fig. 1 illustrates the forma- tion of lipoxins. These compounds can in turn

effect cellular responses which appear to be dis- tinct from those evoked by either leukotrienes or prostaglandins. The responses induced by either

Fig. 4. Skeleton representations. l/4 van der Waals volume representations of the conformers obtained in Table I. Left to right: LXA (I); ll-truns-LXA (I); LXA (c); LXB (C); 8-trans-

LXB; LXB (I).

TABLE I

TORSIONAL ANGLES

Values of the torsional angles after the minimization procedure bearing on all angles.

LXB 8-rrans- LXA 11-trans- LXB LXA

C I I I C I

1 177 180 181 111 63 163 2 358 359 3 3 340 30 3 355 355 359 351 1 341 4 292 294 288 285 294 296 5 215 213 216 214 205 222 6 170 179 180 173 185 149 7 164 163 159 176 174 192 8 177 174 177 174 62 304 9 176 174 356 165 173 211

10 178 178 175 173 52 139 11 181 0 176 182 164 182 12 235 178 111 11 349 179

13 179 180 175 192 174 174 14 62 296 298 197 181 196

15 179 179 185 186 188 195 16 174 174 172 108 122 90

17 177 178 178 187 178 186 18 180 171 178 178 182 191

19 177 174 179 177 168 185

lipoxin A or lipoxin B are stereospecific in that these compounds are more potent than their all- truns isomers, and LXB displays activities which are distinct from those induced by LXA [6-121. In order to gain further insight into the basis for the observed differences in the biological actions of each of these compounds, conformational studies were undertaken with knowledge of the complete stereochemistry of each lipoxin. The molecular structure and the numbering of the torsional an- gles of LXB taken as the initial configuration are illustrated in Fig. 2. Together with the conforma- tion of 8-trans-LXB, Fig. 2 also illustrates the numbering system for a lipoxin-Ca complex. Fig. 3 summarizes the most probable conformations of the four isolated molecules following calculation and energy minimization (these stereo-views are obtained after a simple calculation on the isolated molecules). The spatial conformations generated for LXA (Fig. 3a), ll-rruns-LXA (Fig. 3b), LXB (Fig. 3c), and 8-frans-LXB (Fig. 3d), when com- pared with each other, illustrate that each com-

251

pound possesses a unique spatial conformation with a high probability. As can be seen in Fig. 3,

both LXA and LXB, each of which contains one cis and three truns double bonds in the tetraene conjugation, display a globular structure, while their truns-isomers, which contain four conjugated double bonds (all in the tram configuration), dis- play more rigid extended structures.

It should be noted that each possible complex between a lipoxin and Ca2+ has 27 important

rotational angles. To avoid the large number of conformations that could arise from a systematic analysis of the entire molecule, the procedure was carried out in a stepwise manner on half of the

complexes, and gives one structure tree for each

complex. There is increasing evidence that Ca complexes are symmetrical or quasi-symmetrical [l&20,25-281.

Scheme I shows the structure trees for each lipoxin complex with Ca’+. 9792 and 19872 struc- tures were calculated for each LXB and LXA complex, respectively.

Following application of the energy minimiza- tion procedure [17,21], structure trees degenerate into one or two different complexes. These results

are shown in Table I. Here, I denotes an extended (or interfacial) structure of the complex, while C

denotes a cryptic structure. The globular form is

Fig. 5. Stereoscopic views of the most probable conformations of (LXA),-Ca (full van der Waals volume) in the extended and cryptic

forms.

252

mainly due to many attractive van der Waals interactions, but also to the summation of all the contributions from the energies of transfer in the last minimization step [18,26].

It is clear from the conformations shown in

Fig. 4 (l/4 volume van der Waals) that LXA and LXB may give extended and cryptic structures, the wrapping of LXA around Ca2+ being more pro-

nounced than that of its positional isomer LXB. Finally, Fig. 5 shows the two different conforma- tions of (LXA),-CA given similar conformations,

but the cryptic (C) form is less globular and the equilibrium is in favor of the extended form. Taken together, the results of the present conformational studies performed with the same procedure on four isomeric compounds indicate that they each

lead to considerably different spatial conforma- tions, both in the presence and absence of Ca2+.

The differences between each spatial conforma- tion may account, in part, for the different bio- logical activities of these compounds [6,12] and may provide the basis for their cellular recogm- tion. In a wide range of tissues, the availability of Ca2+ plays a critical role in both the biosynthesis

the and action of eicosanoids [l]. The present results may also provide a conformational basis for interactions between Ca2+ and eicosanoid sub- strate-enzyme complexes, or the interactions be- tween eicosanoids and proteins involved in signal

transduction.

Acknowledgments

R. Brasseur is Chercheur Qualifie at the Fonds

National de la Recherche Scientifique. This work was aided by grant 13-506-867 from the American Heart Association, Massachusetts Affiliate, Inc. (to C.N.S.). C.N.S. is a recipient of the J.V. Satter- field Arthritis Investigator Award from the Arthri-

tis Foundation and a grant from the National Institutes of Health, No. l-R29-GM3876501.

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