In silico modeling of the molecular structure and binding of

leukotriene A4 into leukotriene A4 hydrolase

Paula B. Paz, Esteban G. Vega-Hissi, Mario R. Estrada, Juan C. Garro Martinez*

Area de Qumica Fsica, Departamento de Qumica, Universidad Nacional de San Luis,

Chacabuco 917, San Luis, 5700, Argentina.

*Corresponding author: Tel./Fax (54) 2652 423789 int 122;

E-mail: jcgarro@unsl.edu.ar

Abstract

A combined molecular docking and molecular structure in silico analysis on the

substrate and product of leukotriene A4 hydrolase (LTA4H) was performed. The
molecular structures of the substrate leukotriene A4 (LTA4) and product leukotirene B4
(LTB4) were studied through Density Functional Theory (DFT) calculations at the
B3LYP/6-31+G(d) level of theory in both, gas and condensed phases. The whole LTB4
molecule was divided into three fragments (hydrophobic tail, triene motif, and a polar
acidic group) which were subjected to a full conformational study employing the most
stable conformations of them to build conformers of the complete molecule and
geometry optimize further. LTA4 conformers structures were modeled from the LTB4
minimum energy conformers. Both, protonated and deprotonated species of LTA4 and
LTB4, were analyzed according to pKa values founded in the literature. Finally, a
binding model of LTA4 with LTA4 hydrolase is proposed according to docking results
which show intermolecular interactions that position the protonated and deprotonated
ligand in the active site, in excellent agreement with experimental data.

Running title: In silico modeling of Leukotriene A4.

Keywords: Leukotrienes, Leukotriene A4 hydrolase, Molecular Docking, Binding,
Molecular structure.
Introduction

Leukotrienes (LTs) constitute a family of endogenous metabolites of arachidonic

acid that are biosynthesized via the lipoxygenase pathway (1-3). These interesting
compounds are a class of lipid mediators involved in the development and maintenance
of inflammatory and allergic reactions (4-7). Leukotriene A4 (LTA4), an unstable alkyl
epoxide formed from the immediate precursor 5-HPETE via 5-lipoxygenase (5-LO) (8),
is converted to leukotriene B4 (5S,12R-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoicacid;
LTB4, Figure 1), by stereoselective hydratation of LTA4 hydrolase (LTA4H) (9).
LTB4 is a potent pro-inflammatory mediator implicated in the pathogenesis of a number
of diseases including inflammatory bowel disease (IBD), psoriasis, rheumatoid arthritis
and asthma, and plays an important role in immunological responses, chronic
obstructive pulmonary disease (COPD) and atherosclerosis (4,6,10-12).
On the other hand, LTA4H is a bifunctional zinc metalloenzyme which catalyzes
the rate limiting step in the production of LTB4 (13). The X-ray crystal structures of
LTA4H in complex with different inhibitors have been obtained by several authors (17-
19). According to several investigations, the metal site is located next to the putative
active site bound to His295, His299, and Glu318 (14-18).

In the present paper, we show an analysis of the different interactions of LTA4

into LTA4H through molecular docking study. Molecular and electronic structure
information of LTA4 and LTB4, obtained from an exhaustive conformational analysis,
was used to set an initial conformation for the docking study.
There are few theoretical studies about the molecular structure of LTB4. One of
them was performed by Brasseur at el. using semiempirical methods (19). They present
a computational description of the conformation of a pair of two isomeric molecules (6-
cis and 6-trans-leukotriene B4) forming a complex with one calcium ion. Recently,
Catoire at el. have obtained a highly constrained seahorse conformation when it is
bound to leukotriene receptor BLT2 by 1H-RMN (20). However, there has been no ab
initio and/or DFT studies that analyzes the conformation and electronic structure of
LTA4 and LTB4.
Therefore, we believe that results of this work are of great contribution
especially to elucidate: a) the structural similitude between LTB4 and its precursor
LTA4, b) the conformational changes of LTA4 in gas phase, condensed phase and when
it is bounded to the enzyme, c) the active site of LTA4H where LTA4 binds and
compare it with the interaction site of inhibitors (11-13) and thus, to obtain important
information for future development of new inhibitors of LTB4 formation.

Material and Methods

Conformational study

Relaxed potential energy curves (PECs) were performed at HF/3-21G, HF/6-

31+G(d) and B3LYP/6-31+G(d) levels of theory (21-23), while potential energy
exploratory surfaces (PESs) were carried out at HF/3-21G level of theory. The
conformers were then optimized at a higher level and frequency calculations were
carried out to confirm minimum energy conformers checking the absence of imaginary
frequencies. Solvent effect was evaluated through geometry optimizations using the
integral equation formalism polarizable continuum model (IEF-PCM) (24,25) at
B3LYP/6-31+G(d) level of theory. All calculations were performed using the Gaussian
03 computational program (26).

Molecular Docking

Docking calculations were performed using Autodock Vina software (27) in

order to propose a model of the LTA4-LTA4H complex. The crystal structure of
LTA4H used in our study was obtained from Protein Data Bank (28) (PDB accession
code: 3CHO (19)). The original ligand, 2-amino-N-[4-(phenylmethoxy)phenyl]-
acetamide and water molecules in the crystal structure were removed. LTA4 initial
conformation was obtained from the conformational study. A torsional restriction was
applied for single bonds of the conjugated system of the triene motif fragment setting
them as fixed, i.e., not able to rotate. The box was defined to include completely the
proposed binding site and standard parameters for the docking calculation were used
except for exhaustiveness for which a value of 100 was used.
The lowest binding energy docking complex was analyzed with NCIPlot
program to identify and map noncovalent interactions using the promolecular densities
and its derivatives. These interactions are nonlocal and manifest in real space as low-
gradient isosurfaces with low densities which are interpreted and colored according to
the corresponding values of sign(2) (29).

Results and Discussions

Conformational study for LTB4

To obtain the structure of LTA4 for the docking study, we previously performed
a conformational analysis on LTB4, which optimized structure was used to propose the
initial conformation of LTA4 for the molecular docking study.
Several papers agree that LTB4 is a cis-trans (or E-Z) isomers (5S,12R-
dihydroxy-6Z,8E,10E,14Z-eicosatetraenoicacid) (19,20,30) and that LTB4 show
conformational restrictions in the double bonds configuration of LTB4
(6Z,8E,10E,14Z). Despite the restrictions of double bonds, the molecule of LTB4 has
13 rotational bonds, and if these dihedrals were affected by systematic 120 changes,
more than 1.500x103 conformations could be obtained. To avoid this large number of
possible conformations, a systematic analysis was realized in a stepwise manner on
three different important fragments of the LTB4 easily recognizable: a) hydrophobic tail
b) a triene motif and c) a polar group (Figure 2). Our systematic study includes a severe
analysis of each fragment which was selected based on its chemical characteristics.
Molecular fragmentation have been used in previous work of our group given excellent
results in the study of large molecules (31,32).

Hydrophobic tail
This fragment is composed by a chain of methylens units which is bound to the
triene fragment through a double bond. Conformational study of this fragment consisted
in the analysis of the four dihedral angles presented in Figure 2a. Rotating dihedrals
1 and 4 two potential energy curves were obtained (Figure 3a and b). The plots present
the global energy minimum around 180 for 1 and two local minima next to 120 and
-120 with the same energy for 4. PES of the type E=f(2,3) shows a global minimum
with dihedral values of about 2=180 and 3=180. This is show in supplementary
material, Figure 1S.
 We have found that the hydrophobic tail fragment has high symmetry and an
elongated minimum energy conformation with dihedral values next to 180 for 1, 2, 3,
and 120 or -120 for 4. However, the low rotational barriers (less than 3 kcal.mol -1)
indicate that this fragment has a high conformational flexibility.

Triene motif
Although the conjugated triene system confers the fragment a high rigidity, there
are dihedral angles that deserve to be analyzed: 5, 6 and 7, Figure 2. In contrast to
these dihedrals angles, the values of dihedral 8 depend largely on the fragment with the
acidic function. So, we believe that the analysis of this dihedral has no practical
meaning at this point.
Dihedral angle 5 was studied through a PEC, Figure 3c. The global energy
minimum has the double bond in opposite position respect to the OH group, next to
120, where no interaction occurs, but probably torsional tension is relieved. PES of the
type E=f(6, 7) shown in supporting information as Figure 1S, presents the global
minimum on the region of 6 180 and 7 -120. However, there are local minima
with rotational barriers of about 3 kcal.mol -1 and energy values close to global energy
minimum. The low rotational barrier confers a relatively flexibility, that would only be
restrained in the complete LTB4 molecule by steric repulsion.

Polar (Acidic) group

This fragment is characterized by an OH group over a quiral carbon and a
carboxylic group, both separated by three methylene groups. Due to the methylene
chain, the fragment presents a high flexibility in the dihedral angles 9, 10, 11 and 12. A
similar analysis to the others fragment was performed to the dihedral angles of the
present fragment, briefly: PECs were made for dihedrals 9 and 12, while dihedrals 10
and 11 were analyzed through PES.
In Figure 3d is identified the zone of the highest energy (9 0, repulsion zone)
and zones of minimum energy (9 180, global minimum energy and 9 60, E =
0.1 kcal.mol-1). PES obtained from 10 and 11, plotted in Figure 1S, shows that the
global energy minimum is localized next to 60 for both, 10 and 11. The low rotational
barriers between any of the two minima indicate the high flexibility of the fragment.
 Furthermore, rotation of dihedrals 12 and 13 produces a series of conformers
with intramolecular hydrogen bond interactions. Consequently, a NBO analysis was
performed on these conformations. In addition, taking into consideration the values of
pKa for leukotrienes range from 3 to 5 (33,34), deprotonated species should also be
studied. Table 1 presents the three potential conformations for acidic group in both
species obtained from DFT calculations. The total energy indicates that both species of
the first conformer are the most stable conformations. The interaction energy E(2)
between donor and acceptor orbitals show an effective interaction between the oxygen
lone pairs (LP) and the sigma antibonding orbitals (*) of OH bond which is higher in
the deprotonated species (no charge transfer interaction was founded in the last
conformer for the anionic species). Although the third acidic conformer presents a
higher charge transfer stabilization energy, steric restraints could disfavor this
conformation.

Molecular structure for LTB4

Using the minimum energy conformers of the three fragments we proposed an
initial molecular structure for LTB4. However, its stable conformation could be the
same or different to structure proposed. Figure 4 shows the superposition of the most
stable conformer and molecular structure obtained from the fragmentary study. The
principal difference of both structures is the values of dihedral angle 8. The value
proposed for 8 in the initial structure was of 180 (Figure 4, in dark gray) while full
optimized value is -125 (Figure 4, in light gray) giving rise to a E between both
structures of about 35 kcal.mol-1. This result is expected because dihedral 8 was not
analyzed in the fragmentary analysis. Consequently, dihedral value was modified when
the whole molecule was fully optimized.

Molecular structure for LTA4

LTB4 shows a high structural similarity to its precursor the leukotriene A4. The
most important structural difference resides in the presence of an epoxy group in LTA4
molecule. This epoxy group is hydrolyzed and converted into an OH group in the
enzymatic step that involves the formation of LTB4 (13-18).
 To obtain a stable conformation for LTA4, which can be used in molecular
docking study, we suggested an initial conformation using the dihedral values of LTB4.
Hydrophobic tail dihedral initial values were taken from the most stable conformation
of LTB4. In addition, literature suggests a 7E, 9E, 11Z, 14Z configuration for the
unsaturated part of LTA4 (8). However, dihedrals 1 and 2 of LTA4, Figure 5, are
involved in the orientation of the epoxy group which plays a key role in enzyme
binding. Consequently, to perform a complete analysis, these important dihedrals were
studied.

A full optimization carried out for the four possible conformers gave low energy
differences between all conformers, Figure 2S in supporting information. Solvent effect
analysis indicates that the stable conformations obtained in vacuo are not altered by the
presence of the solvent.

Molecular Docking
A molecular docking study was performed to propose a binding model and
interpret the different interactions between the ligand and the enzyme. Although the
reaction mechanism proposed by Thunnissen et al. (14) involves the deprotonated
species of LTA4, both, the acidic and anionic species of LTA4, were subjected to
docking studies.
Nine possible binding complexes were obtained for the anionic species of LTA4.
The most stable one, i.e. the one with lowest G of binding (about -9.5 kcal.mol-1), is
shown in Figure 6. The model reveals that the carboxylate group interacts with Arg563
and is close to Lys565; the epoxide is positioned with the oxygen atom coordinating
with Zn2+ cation and forming a square based pyramidal metal complex with His 295,
His 299 and Glu 318 (14); the hydrophobic tail is buried in a hydrophobic pocket; and
Asp375 is oriented toward the triene group where it would control the position of a
water molecule and therefore the stereospecific insertion of the 12R-hydroxyl group of
LTB4 as is proposed in the literature according to the reaction mechanism presented
(14-18).

The most stable docking complex of the enzyme and the acidic species of LTA4
presents the substrate located in the same cavity and with almost the same orientation
that was founded for the anionic one (Figure 6). Moreover, G of binding is only 0.1
kcal.mol-1 higher.
Figure 6 also shows a complex web of weak noncovalent interactions between
the substrate and the active site of the enzyme (including the Zn 2+ cation) where van der
Waals and hydrophobic interactions dominate by far (green surfaces). Moreover,
interaction analysis reveals a hydrogen bond between the carboxylate group of LTA4
and Arg563. In the proposed binding model LTA4 do not interact with Lys565.
However, we expect that the dynamic behavior of both, the substrate and the enzyme,
leads to the formation of a hydrogen bond (or more probably a salt bridge) between
LTA4 and the positively charged residue Lys565 due to it is close to Arg563 in the
binding site.
LTA4 minimum energy conformation from quantum analysis shows differences
with enzyme-bounded conformation (Figure 7). However, it is well known that flexible
molecules change their conformation upon binding to a protein (35), thus, it is not
surprising our finding if we take into account the high degree of flexibility of LTA4
molecule.

Conclusions

We obtained from conformational analysis the stable structures for the substrate
and product of the enzyme LTA4H using Hartree Fock and DFT calculations. Most
stable conformation of LTB4 shows an elongated structure with an intramolecular
interaction in the polar fragment analyzed through NBO analysis. Based in the structural
data of LTB4 four possible conformations of LTA4 were proposed. We founded that
conformer IV is the most stable in gas and solvated phases with a E of 1 kcal.mol-1 less
than the other conformers.
Molecular docking results provided an interaction model of LTA4 with LTA4H
in excellent correlation with the experimental data: the substrate fits in the active site of
the enzyme arranging its functional groups according to the reaction mechanism
proposed by several authors (14-17). Both species of LTA4, protonated and
deprotonated, bind to the same site with proper orientation of the key groups for the
enzymatic reaction. The substrate is held in the active site by multiple weak attractive
interactions along its surface and a hydrogen bond to Arg563.
 Thus, the in silico model of LTA4-LTA4H complex obtained from molecular
docking in combination with a conformation analysis of substrate and product provides
insights into the development of new and most efficient inhibitors of LTB4 formation.

Acknowledgements
This work was supported by Consejo Nacional de Investigaciones Cientficas y
Tcnicas (CONICET) project PIP11220100100151 and Universidad Nacional de San
Luis (UNSL).
Figure Legends

Figure 1: Reaction catalyzed by LTA4H. Chemical structures of LTA4 and LTB4.

Figure 2: Structure of LTB4 (5S,12R-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoicacid).

Scheme of fragments of LTB4 and dihedral studies: a) hydrophobic tail, b) triene motif,
c) polar group.
Figure 3: Curves energy potential for; a) and b) hydrophobic tail, c) triene motif and d)
acidic part.

Figure 4: Overlap of the most stable conformer of LTB4 (light gray sticks) and
molecular structure obtained from the fragmentary study (dark gray sticks).
Figure 5: Molecular structure for LTA4 (5S,6S-epoxy-7E,9E,11Z,14Z-eicosatetraenoic
acid).

Figure 6: Model of LTA4 anion bounded to LTA4H. Docked complex showing the
side chains of the amino acids involved in the reaction mechanism proposed (left panel).
Combined docking results of acidic and anionic species of LTA4 (upper right panel).
Noncovalent interactions of the LTA4 anion within the binding pocket of LTA4H
(lower right panel). Blue indicates strong attractive interactions and green van der
Waals (weak attractive) interactions.
Figure 7: Overlap of the most stable conformer of LTA4 (dark gray sticks) and
molecular structure obtained from the molecular docking study (light gray sticks).
 Table1: Conformational and electronic dates obtained by rotation of dihedrals 12 and
13 for polar fragment.
Form 12 13 E(2)a Total Energyb E
-1
kcal.mol (Hartree) kcal.mol-1
LP(1) 2.06
Protonated -77.70 -178.70 -461.5722022 0.0
LP(2) 5.34

LP(1) 6.98
Deprotonated -77.84 - -461.0349725
LP(2) 34.67

Protonated 86.96 179.52 LP(1) 2.92 -461.5698006 1.50

LP(1) 6.98
Deprotonated 100.69 - -461.0349725
LP(2) 34.67

Protonated 94.72 3.01 LP(2)16.83 -461.5702739 1.21

Deprotonated 15.06 - Not founded -461.0181532

a
E(2) means energy of hyper conjugative interaction (stabilization energy). LP: Lone Pair
b
Total energy of conformer in Hartree (1hartree=627.5095 kcal.mol-1).

Table 2: Energy and structural dates for the conformers of LTA4.

HF/6-31+G(d) B3LYP/6-31+G(d) IEF-PCM/ B3LYP/6-31+G(d)

E E E
1 2 1 2 1 2
(kcal.mol-1) (kcal.mol-1) (kcal.mol-1)
I -72.58 93.46 2.25 -66.52 92.92 2.33 -63.82 93.57 2.19
II -73.72 -148.45 0.62 -67.36 -147.62 1.00 -64.25 -152.00 1.65
III 148.23 92.08 1.69 151.11 91.49 1.40 153.48 91.35 0.60
IV 148.03 -149.07 0 150.28 -149.90 0 154.00 -153.16 0
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Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure 1S: Potential energy surfaces: A) E=f(3,4) hydrophobic tail, B) E=f(6,7)

triene motif, C) E=f(10,11) polar group.

Figure 2S: Stable conformers for LTA4 at B3LYP/6-31+G(d) levels of theory.

 A B
Global minimun

Global minimun

Global minimum

Figure 1S: Potential energy surfaces: A) E=f(3,4) hydrophobic tail, B) E=f(6,7)

triene motif, C) E=f(10,11) polar group.
 Conformer I Conformer II

Conformer III Conformer IV

Figure 2S: Stable conformers for LTA4 at B3LYP/6-31+G(d) levels of theory.