PROTEINS:Structure, Function, and Genetics 14:45-64 (1992)

Modeling of Substrate and Inhibitor Binding to Phospholipase A2 Richard B. Sessions, Pnina Dauber-Osguthorpe,Malcolm M. Campbell, and David J. Osguthorpe Molecular Graphics Unit, University of Bath, Bath BA2 7AY, United Kingdom

ABSTRACT Molecular graphics and molecular mechanics techniques have been used to study the mode of ligand binding and mechanism of action of the enzyme phospholipase A,. A substrate-enzyme complex was constructed based on the crystal structure of the apoenzyme. The complex was minimized to relieve initial strain, and the structural and energetic features of the resultant complex analyzed in detail, at the molecular and residue level. The minimized complex was then used as a basis for examining the action of the enzyme on modified substrates, binding of inhibitors to the enzyme, and possible reaction intermediate complexes. The model is compatible with the suggested mechanism of hydrolysis and with experimental data about stereoselectivity,efficiency of hydrolysis of modified substrates, and inhibitor potency. In conclusion, the model can be used as a tool in evaluating new ligands as possible substrates and in the rational design of inhibitors, for the therapeutic treatment of diseases such as rheumatoid arthritis, atherosclerosis, and asthma. o 1992 Wiley-Liss, Inc. Key words: proteins, active site, computer simulations, molecular mechanics, molecular graphics, structure, binding energies INTRODUCTION PLA, catalyzes the hydrolysis of the sn-2 acyl side chains of phosphoglycerides. The enzyme is responsible for liberating arachidonic acid from the membrane pool, which is a precursor of inflammatory mediators such as prostaglandins, prostacyclins, and leukotrienes. There is, therefore a growing interest in designing inhibitors of this enzyme as its control could provide therapeutic treatment of diseases such as rheumatoid arthritis, atherosclerosis, and asthma. The objective of this study was to understand the mode of binding and the hydrolysis mechanism in terms of structure and energy, a t the molecular level. This understanding should facilitate a rational design of substrate analogues or inhibitors. Since there were no experimental structures of the enzyme and a substrate (or inhibitor) when we began the modeling, we utilized as much as possible other available experimental data in de0 1992 WILEY-LISS, INC.

signing and testing the model for the complex. The X-ray structures of PLA, from a number of sources have been determined snake venom as a calcium free dimer,' porcine pancreatic as the apoenzyme,2 and as a m ~ t a n t ,bovine ~ pancreatic as the , ~ with an in~ y m o g e nand , ~ as the a p ~ e n z y m eand hibitor covalently bound to the active site histidine.6 The venom and pancreatic structures have been closely ~ o m p a r e dThe . ~ X-ray structures show high structural homology, especially in the active site region. The human pancreatic enzyme has a high sequence homology compared with the bovine pancreatic phospholipase A, (80% identity).' There is immunological evidence for similarity between extracellular and intracellular membrane associated PLA,.' Sequences have been recently determined of a membrane associated rat platelet PLA," and from human rheumatoid arthritic synovial fluid.'' Both of these show strong sequence homology with the venom phospholipases (Type-11).The N-terminal sequence of PLA, from human placental membranes also suggests that this belongs to the Type-I1class.12 All of this information indicates that inhibitors designed for the extracellular PLA, are likely to be active upon intracellular, membrane-associated, and inflammatory phospholipases implicated in the disease processes of interest. From the X-ray structures of the enzyme a cleft can clearly be seen; most residues lining the cleft are hydrophobic except for the important His-48 in the deepest part of the cleft. The enzyme is rapidly methylated on N6 of this His by methyl-p-nitrobenzenesulfonate. This results in complete inactivation of the enzyme while substrate and calcium binding are hardly affected.13 The crystal structure of the enzyme also reveals that this His residue is hydrogen bonded to a well-determined structural water molecule and to an aspartic acid residue (Asp-99) in a couple reminiscent of that found in serine proteases. Therefore, it has been proposed that the

Received January 25,1991; revision accepted June 21,1991. Address reprint requests to Dr. David J . Osguthorpe, Molecular Graphics Unit, School of Chemistry, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom. Present address of Richard B. Sessions: Department of Biochemistry, Medical School, University Walk, University of Bristol, Bristol, BS8 lTS, United Kingdom.

46

R.B. SESSIONS ET AL.

mechanism of catalysis is analogous, except that the nucleophile is the water molecule rather than a serine OH, such that the first step of the reaction produces the tetrahedral intermediate of ester hydrolysis directly, rather than the covalent subs t r a t e n z y m e complex seen in the serine proteases. PLA,s have an absolute requirement for calcium for activity. The calcium is fairly weakly bound to the protein (K, ca. mM) in a highly conserved loop in the active site cleft.14Inspection of the crystal structure of bovine PLA,13 also suggested that the ester carbonyl function might be polarized and hence activated, both by coordination to the calcium ion and by hydrogen bonding to the backbone N-H of Gly30. Laser-induced europium luminescense spectroscopy before and after binding of the substrate analogue n-decyl phosphocholine indicated that about one molecule of water was displaced from the metal on complexation of monomeric substrate, presumably by coordination of the phosphate to the metal." This experiment implied that the phosphate might coordinate the calcium in the native substrate enzyme-calcium-substrate complex. In this study we derive a model for ligand binding to PLA, based on the crystal structure of the extracellular apoenzyme' using molecular graphics and molecular mechanics modeling techniques. Preliminary results describing a substrat+enzyme complex were published earlier.16.17 The compatibility of this model with experimental data is examined. In addition, partitioning of the energy provides an insight into the nature of the ligand-enzyme interactions. The model provides a basis for examining fundamental questions such as the nature of the hydrolysis transition state, explaining the basis of the enzyme's stereospecificity and evaluating the likelihood of proposed ligands being effective inhibitors.

METHODS The molecular mechanics calculations used in this study are based on representing the energy of a molecular system as an analytical function of internal coordinates and interatomic distances.

The first four terms represent the energy required to distort the internals from their ideal values: b, 8, and x are the bonds, valence angles, torsion angles, and out of plane angles respectively; b, and 8, are the ideal bond lengths and angles; and the Hs are the force constants for distorting these internals. The last terms represent the interaction between

+,

nonbonded atoms with r* being the most favorable interatomic distance and E the energy of interaction at this distance, and qi are the partial atomic charges. The force constants were determined by fitting structural and spectroscopic properties of small model compounds." Given a n analytical representation of the energy of a molecule, V, the energy of the system is minimized by solving the equations:

aV/axi= 0,

i = 1, 312

(2)

where xi are the Cartesian coordinates of the atoms in the system and n is the number of atoms. The protocol for setting up the molecular systems and minimizing the energy was designed to minimize disruption of the overall structure due to local strain. The highest resolution X-ray structure of bovine pancreatic PLA, was used as the basis for modeling the enzyme-ligand interactions. First, the apoenzyme system was set up. The experimental structure provided initial positions for the heavy atoms of the protein, the calcium ion, and water oxygens. Further water molecules were generated to fully solvate the protein up to a distance of 3.5 A from the surface. Of these observed and generated waters only a subset was kept. (1) The active site was fully solvated since this is the focus of our investigation-water molecules up to 10 A away from His-48 were maintained; (2) to provide shielding of the electrostatic interactions of charged residues, water molecules up to 3.5 from any of the charged groups were kept; and (3) bridging waters which link (by hydrogen bonds) a t least two residues were retained since they are important for the integrity of the structure of the protein. Hydrogen atoms were added to the protein consistent with a pH of 7.0. The hydrogens of water molecules were added in orientations which optimize interactions with close protein residues. The minimization was carried out in stages. First we performed minimizations in which the protein heavy atoms and oxygens of crystallographic water molecules were constrained to their experimental positions by a penalty function. This enabled relaxation of all built atoms without significant disruption of the overall protein structure. Then all constraints were removed and minimization was performed using a steepest descent algorithm to remove any remaining large strain. Finally nonconstrained minimizations were performed using a conjugate gradient method until convergence was achieved (average derivative smaller 0.01 kcal mo1-l A-'). Complexes of the protein and various ligands were built by docking the ligand into the structure of the minimized apoenzyme (see below). This was followed by a constrained minimization keeping all protein heavy atoms tethered to relive initial bad interactions, and then a full unconstrained minimization until convergence.

MODELING OF LIGAND BINDING TO PLA,

Molecular dynamics simulations were carried out on the apoenzyme and substratenzyme complex. The simulations were performed by solving Newton’s equations of motion:

Fi= m,u,

Fi= avlax,

i=1,3n.

(3)

These equations were solved numerically using a leap-frog algorithm, with a time step of 1fsec. Initial coordinates for the simulations were obtained from the corresponding minimized structures. Random initial velocities with a Boltzmann distribution consistent with 300 K were assigned to the atoms. The system was allowed to equilibrate for 1psec using a thermal bath. In the remainder of the simulation (51 and 55 psec) the thermal bath was not required since the average temperature remained constant. The trajectory was analyzed using the program FOCUS.1S

RESULTS AND DISCUSSION Minimized Apoenzyme The minimized apoenzyme showed high structural similarity to the crystal structure. The rms fit of all heavy atoms is 1.25 A while that of just the main chain heavy atoms is 1.07 A, which indicates greater movement of the side chains during minimization. Likewise the rms fit of just the secondary structure main chain heavy atoms is 0.95 A indicating greater movement in the loop regions, as illustrated in Figure l a , the plot of rms per residue. This is in general agreement with the temperature factors of the corresponding atoms or segments in the crystal structure. In a molecular dynamics study of the apoenzyme similar phenomena were observed, i.e., backbone atoms move less than side chain atoms and secondary structure elements are less mobile than intervening loops.2o The scatter plot in Figure l b shows the rms deviation compared with surface exposure for each residue. The percent surface exposure was determined approximately by dividing the surface area of the residue in the protein by that of the residue in an extended conformation in a strand of polyglycine, (the surface areas were measured using Connolly’s algorithm). A general correlation between rms and surface exposure can be discerned. Hence, while the rms deviations of the 11 completely buried residues lie between 0.3 and 0.8 the two residues with an rms greater than 2.2 (Ser-34 and Lys-121) are almost completely exposed to solvent (=go%). Lipid Conformations Several crystal structures of phospholipids have been rep~rted.’~-’~ (See Diagram I for a schematic representation and nomenclature of chains and torsion angles for phospholipids.) All the crystal structures feature extended (staggered) alkyl chains lying parallel to one another; this arrangement is

47

achieved by a gauche interaction in the ~ 2 x tor3 sion of either the sn-1 (y4) or sn-2 (&) chain. The coordinates of two structures have been deposited in the Cambridge Structural Database (CSD), and these represent both types of kink described above. The most common conformation observed in the crystal, solution, and membrane environments has the kink in the sn-2 chain.25

Substrate Docking Into the Apoenzyme We attempted to dock the substrate into the active site of the minimized bovine PLA, structure in a fashion consistent with the constraints provided by the mechanistic proposals and experimental data. The procedure was carried out manually on a graphics display system, with the least possible modifications to the experimentally observed conformations of the isolated lipid and apoenzyme. Two initial lipid conformations were provided by the structures in the CSD. The conformation with the kink in the sn-1 chain could not be docked in any reasonable manner, whereas that with the kink in sn-2 could be fitted after some modifications to the glycerophosphate torsions, which are detailed in Table I. These modifications essentially introduce a gauche interaction at the Cl-O(P) position (al), a rotamer that is significantly populated in solution.26 During the docking procedure it was also necessary to move four side chains of the protein to enable a good fit and orientation of the reactive groups. Leu-19 x1 was altered from -63 to 66 and xz from 176 to 88 in order that the sn-2 chain could pack closely against the wall of the hydrophobic pocket in that region. Leu31 and Tyr-69 were nearly in contact across the entrance of the active site cleft in the minimized structure and these were rotated out of the way of the sn-1 chain (Leu-31 x1 from -67 to -78, x, from 169 to 173; Tyr-69 x1 from -60 to 58, x, from 136 to 84). At this point the diacylglyceride part of the model substrate occupied a large proportion of the active site cleft and the ester function of the sn-2 chain approximately lined up with the His-48 and its hydrogen bonded water molecule. It appeared possible to coordinate the phosphate of the phosphocholine to the calcium as well by displacing the carboxylate of Asp-49 from the first coordination sphere of the calcium. At the time this modification was supported by experimental data. An enzyme with Lys replacing Asp-49 appeared to be active, although the calcium ion was bound after the substrateenzyme complex was f ~ r m e d . ’The ~ ~ ~ethanolamine ~ side chain was given a staggered conformation, but as it protrudes into solvent it is free to adopt gauche conformations which are energetically more favorable.26Examination of a5 in Table I shows that this gauche conformation is adopted in the inhibitor complex. The presence of this side chain is not required for PLA, to perform its catalytic function.” After achieving a good “manual” docking of the sub-

48

R.B. SESSIONS ET AL.

RMS per Residue RMS

..

3 2.5

-

21 C -

1 -

0 6 -

0

--

23

0

-I

A

30

40

C

B

Alpha helix

80

120

100

140

residue number

==-

Q

E

Beta sheet

m

F

RMS vs YO exposure to solvent

b

% exposure 1 ic

80

. .-. -. ._- - . . .

,

~

.

..-

+..

.*

60 40-

.a

.

20 0 0.

.-

- >-. -1 ..

B?-.-3

=

*

t .

. -. - .

05

..

*

.

*

. ---

1

15

2

2.5

3

RMS

Fig. 1. Rms deviation of the minimized structure of the apoenzyme from the crystal structure. (a) Rms deviation of each residue. (b)Rms deviation as a function of surface exposure of the residues.

strate, the complex was minimized until convergence (see Methods).

EnzymG3ubstrat.eComplexes Structural aspects Minimization of the energy of the ternary complex achieved a relaxed structure, while retaining overall structural similarity to the initial conformations

of the enzyme and lipid, and preserving all the salient features of the proposed catalytic mechanism. The overall rms deviation between all backbone atoms of the apoenzyme and the corresponding atoms in the complex is ~ 0 . A. 6 The corresponding deviation for all heavy atom is ~ 0 . A. 9 Most of these deviations originate from the initial adjustments in side chain conformation of some of the residues in

49

MODELING OF LIGAND BINDING TO PLA,

TABLE I. Lipid Torsions* Complex LipidX-ray -52 Substrate-A initial 56 Substrate-A 71 Substrate-B 72 Substrate-C 76 Enantiomer -14 Protonrelay 79 Charge separation 82 Inhibitor 71

8,

e3

65 -172

64

a1

69 154

a,

58

177 175 55 28 -165 -170 173 53 83 -177 -170 173 53 83 -177 -161 -146 89 35 158 -137 -139 -48 104 166 -158 176 55 46 172 -158 -172 -168 177

66 56

70 90

a3

66

a4

106

a5

67

PI 97

174 -175 -177 95 155 -164 179 94 155 -164 179 94 150 57 178 118 167 -98 -171 -115 168 -71 -173 85

178 145 135 167

-82 -91

-166 68

68 61

PZ

P3

179 -119 -172 -177 -177 172 -177 178 171 158

*For the definition of the torsions see Diagram I. Torsions are given in degrees.

P4

PS

YI

65 -178 -178

YZ

73

173

179 -171

~4

YS

-173

-108 68 169 -162 164 176 175 -178 -110 61 178 118 -165 -158 178 180 -110 61 177 118 -165 -158 178 180 -137 61 -178 -111 177 140 -169 -179 128 -60 -177 -95 179 118 179 -179 -82 65 172 -178 178 144 174 176 -63 -80

72 67

164 174

176 74

160 171

166 178

175 176

177 178

50

R.B.SESSIONS ET AL.

a

b

Fig. 2a,b. Legend appears on page 51.

the active site in order to enable ligand docking. Leu-19, Leu-31, Asp-49, and Tyr-69 deviate by 2.4, 1.5, 2.0, and 3.6 A, respectively. Rearranging the side chain of Leu-19 away from the region where the lipid was being docked left some “extra” space next to the ligand. Consequently the following residues, 20-23, moved closer to optimize the interactions with the lipid. (Thus these residues also exhibit a large rms deviation 1.5-2.2 A.) Similarly Leu4 (rms= 1.8A) moved significantly in order to achieve better interactions with the lipid. Active site structure. The structure of the active site region in the minimized complex is shown in Figure 2a. All protein residues and water molecules within 3.0 A of a lipid atom o r the calcium ion and a few other key residues-His-48, Asp-99, and Tyr-69 are shown. The hydrolysis reaction center main-

tained all the features consistent with the suggested mechanism. The catalytic couple His-48-Asp-99 interacts through a -N‘-OY hydrogen bond. A catalytic water is hydrogen bonded to the N’ of His-48, and is 3.18 b from the carbonyl carbon of the ester, poised on the beginning of the reaction coordinate. The oxygen of this water molecule has only one hydrogen bond (to another water molecule), while the second lone pair which could form a hydrogen bond is oriented toward the reaction center. The angle made by the water oxygen, carbonyl carbon, and carbony1 oxygen is -95” in accordance with the observation that during the addition of nucleophiles to carbonyl moieties this angle should be greater than In addition, the carbonyl oxygen of the ester function is 2.7 A from the calcium ion and 1.9 h; from the N-H of Gly-30. Both of these interactions PO-

MODELING OF LIGAND BINDING TO PLA,

51

C

d

h

R

Fig. 2. Stereo picture of the active site of the minimized substrateenzyme complexes (a-c)and of the "wrong"enantiomer of the substrate (d). Ligand bonds are represented by filled bonds. Hydrogen bonds are represented by dashed or dotted lines (for

dw. c 2.2 A and 2.2 5 dH.. 5 2.6 A, respectively). The calcium ion is indicated by + + " and distances to coordinatedatoms are marked by dashed and dotted lines.

larize the carbonyl and thus activate it toward nucleophilic attack. The phosphate group of the lipid is stabilized by interactions of two oxygens with the calcium ion, and hydrogen bonding to the NH of Gly32. All other polar groups of the substrate the sn-1 carbonyl, the sn-2 ester oxygen, the phosphate oxygens, and the amine group are stabilized by interactions with solvent molecules. All of the other close interatomic distances are to nonpolar groups of the residues lining the cavity-Leu-2, Phe-5, Asn-6, Leu-19, Phe-22, Asn-23, Cys-29, Gly-30, Leu-31, Gly-32, and Cys-45. As mentioned above, to enable docking it was necessary to move the side chains of some of the residues out of the way. However, due to readjustments of backbone positions during the minimization some of these side chains regained their initial relative

orientation, while others could be rotated back manually. During the minimization the side chain of Leu-31 had rotated back so that the final torsion was similar to that of the starting conformation. Those of Leu-19 and Tyr-69, however, were rotated back to xls similar to the initial values without incurring bad clashes with protein or lipid. Minimization of this refined model rapidly converged to that shown in Figure 2b. The rms deviation of the main chains of these two complexes is 0.09 A, and the overall rms of all heavy atoms is 0.43 A. This overall difference originates mainly from the deviations in side chain positions of Leu-19 and Tyr-69 (2.2 and 3.2 A, respectively). All other residues deviate less than 0.5 A. This modification enabled more hydrophobic contact between Leu-19 and the sn-2 side chain and brought the hydroxyl group of Tyr-69 into hydrogen

"

52

R.B. SESSIONS ET AL.

bonding contact with the ester carbonyl of the sn-1 chain. The Tyr-69 hydroxyl also interacts with the phosphate of the substrate via hydrogen bonding involving two intervening waters. The flexibility of some of the side chains in the active site region implied that some variation in the details of the model is possible. We investigated additional possible orientations of the side chains of Tyr-69 and Asp-49. Adjustments of the side chain torsions of the Tyr residue can bring the hydroxyl to within hydrogen bonding distance of the lipid’s phosphate group. In addition it is also possible to bring back the side chain of Asp-49 to within coordination distance of the calcium ion. The minimized structure of this complex is shown in Figure 2c. This complex is consistent with recent experiments that suggested that the Tyr-69 is important for the stereospecificity of the enzyme, since replacement of Tyr-69 by Phe causes the protein to lose some of its stereo~pecificity.~~ The interactions between this residue and the phosphate may be important in ensuring that the L isomer is bound in an appropriate orientation for reaction (see below). This model has many similarities to a crystal structure of PLA, complexed to a substrate derived inhibitor which was published while preparing this man~script.~’ The similarities include the polarization of the carbony1 by the calcium ion and the NH of Gly-30, coordination of the calcium to Asp-49 side chain and to the phosphate of the ligand, and the hydrogen bonding between the ligand’s phosphate and the OH of Tyr-69. Calcium coordination geometry. In the apoenzyme the calcium ion is coordinated by four protein residues and 3 water molecules. Three of the residues involve the loop 28-32, with the carbonyls of alternate residues (Tyr-28, Gly-30, and Gly-32) pointing toward the calcium. The fourth residue is Asp-49, which has its charged side chain pointing toward the calcium. In the subtrate-enzyme complexes A and B the interactions with the loop 28-32 were maintained, and in addition the two phosphate oxygens are pointing toward the calcium. Although the side chain of the Asp-49 was rotated to enable lipid docking it is still close enough (3.8 A) to interact favorably with the calcium (see below). In complex C the environment of the calcium is more similer to that in the original apoenzyme, with coordination to only one phosphate oxygen and to the Asp-49 side chain (see Fig. 2c). The coordination chemistry of calcium in crystal structures has been examined and many examples of 6, 7, and 8 coordination numbers have been observed with a wide variety of associated coordination g e ~ m e t r i e sAs . ~ might ~ be expected from its noble-gas electron configuration the cation, like the other alkali and alkaline earth metal ions, shows no particular directional preference for coordinated ligands (unlike transition metals). A search of the

CSD showed that there are currently 12 high-resolution small molecule structures solved which contain both calcium and phosphatetphosphonate ions. In each of these structures every phosphate is coordinated to a calcium ion. The most common type of coordination is a close contact between a single oxygen of a phosphate and the metal. In the sole example of a phosphate calcium interaction in the Brookhaven database the coordination is of this monodentate type.34There are, however, several examples of a “chelate” interaction between the phosphate and the calcium, i.e., two oxygens of one phosphate are in close contact with the metal. This interaction is accompanied by a strained (smaller than average) 0-P-0 valence angle to enable both oxygens to be as close as possible to the calcium. For example the 0-P-0 angle in one such structure35 is 98”, which is similar to the corresponding angles in substrate-A and -B complexes ( ~ 9 0 ” )The . lack of a strong preference for monodentate versus chelate geometry is reflected in this set of minimizations, where one or two phosphate oxygens can be coordinated to the calcium. It is noteworthy that both in the substrate-C model, where only one phosphate oxygen coordinates the calcium, and in those complexes which became similarly monodentate on minimization (proton relay complex and phosphonate inhibitor, below) it is the same prochiral oxygen which coordinates the metal. The R, and S, isomers of lecithins with a sulfur present on the phosphate have been synthesized and their reactivity tested against porcine pancreatic PLA,.31 Only the enantiomer is hydrolyzed by the enzyme. The stereochemistry in terms of our model (substrate C) is such that the phosphate oxygen is coordinating the calcium and the sulfur hydrogen bonds to the Tyr-69 phenolic OH, while binding the S, isomer would imply coordination of the sulfur to the calcium. Since the calcium ion has a strong preference for coordinating the oxygen over the sulfur only the R, will be hydrolyzed.

Energetic aspects The intra- and intermolecular energies of the apoenzyme and the enzyme-ligand complexes are summarized in Table 11. The intermolecular energies are partitioned into molecule-molecule interactions in Table I11 and to ligand-residue and calcium-residue interactions in Tables IV and V, respectively. Comparing the intramolecular energies of the apoenzyme with the corresponding energies of the protein in the three enzyme-substrate complexes reveals that ligand binding has induced some internal strain in the protein. This is manifested in a higher valence energy in all complexes and a higher intramolecular van der Waals energy for complexes B and C. The intramolecular Coulomb energy of the protein in complex C is also higher than that of the

53

MODELING OF LIGAND BINDING TO PLA,

TABLE 11. Total Inter- and Intramolecular Energies of the Complexes*

Complex Apoenzyme

Molecule Protein Calcium Water Total

Valence 1205 136 1341

Protein Calcium Lipid Water Total

1213

Substrate-A

Substrate-B

Substrate-C

Enantiomer

Charge-separation

Inhibitor

Total 504

515

-1216

136 640

Intermolecular Coul Total - 1415 - 1464 13 -325 -313 460 -3579 -3118 424 -5319 -4895

515

-

443

-

1285

vdW -49

- 1397

52 10 -15 413 356

- 1404

1456 -303 -226 -2880 -4865

- 1005

-313 -211 -3293 -5221 -1639 -322 - 185 -7189 -9335

-1770 -310 -206 -6177 -8463

-1147 -310 -205 -5781 - 7443

-

1410 -308 -203 -3371 -5292 - 1415 -399 -345 -3368 -5527

-

10 - 12 316 372 -50 13 -9 443 397

1464 -298 -215 - 3055 -4920 - 1464 -385 -354 -2927 -5130

1009 -298 -203 -2934 -4332 -966 -385 -368 -2805 -4524

-52 12 -7 423 376

-1413 -395 -367 -3347 - 5522

- 1465 -383

-

-374 -2924 -5146

-372 -2802 -4621

-52 13 - 12 446 395

- 1435 -403

- 1487

1093 -390 -325 -2829 -4637 ~. .

9

- 38

524

- 1323

9 121 573

Protein Calcium Lipid Water Total

1214

522

-

1285

451

38 116 1368

11

-37

533

-1322

12 116 579

Protein Calcium Lipid Water Total

1222

523

-1121

624

33 395 1651

14

-46

536

-1168

-1 395 1019

-131 12 -21 1012 872

1222

520

-1287

455

-54

Protein Calcium Lipid Water Total Protein Calcium Lipid Water Total

46 121 1389 1224

9 529 509

-

-

43

1320

- 1235

12 121 588 498

24 122 1370

528

-

1292

14 122 606

1219

522

- 1340

401

19

-57

-

42 122 1383

537

-1396

2 122 525

1234

519

- 1359

394

15

28 125

18

1387

537

56

-

-68 - 1427

-22 125 497

Total -960 -313 -2982 -4255

54 11 - 12 424 369

38 121 1372

Protein Calcium Lipid Water Total Protein Calcium Lipid Water Total

Proton-relay

Intramolecular vdW Coul -1216 515

-

-316 -212 -3358 -5283

-291 -3400 -5529

-1451 -305 -224 -2934 -4914 -

-390 -303 -2954 -5134

- 1008

-305 -215 -2813 -4341 -303 -214 -2764 -4286

-

1064

- 383

-

*Energies are in kcal/mol.

apoenzyme. In contrast, this energy is more favorable in complexes A and B. This can be traced mainly to interactions of Asp-49 with the charged residues Arg-43 and Lys-56. As mentioned before, the side chain of the Asp residue was oriented toward the calcium in the experimental structure and was moved to enable ligand binding. This reorientation resulted in a reduction in the protein-calcium interaction (see Table 111). On the other hand, the Asp side chain was brought closer to the side chains

of Arg-43 and Lys-56. Although there is no direct contact between these residues, the electrostatic interactions are significantly more favorable. As seen from Table I11 ligand binding significantly reduced the protein-calcium and calciumwater interactions. Water-water interactions are reduced as well; however, this mainly reflects the fact that many water molecules have been replaced by the ligand. (The water-water interaction in complex C is higher due to a larger solvation shell in this

54

R.B.SESSIONS ET AL. TABLE 111. Intermolecular Energies* Proteinlipid

Complex Apoenzyme Substrate-A Substrate-B Substrate-C Enantiomer Proton-relay Charge-separation Inhibitor

Proteincalcium -424.1 -325.3 -325.5 -393.1 -335.6 -333.1 -302.4 -341.1

-65.7 -75.1 -109.7 -67.8 - 102.0 - 127.9 -99.9

Lipidcalcium

Lipidwater

-240.6 -238.8 -165.1 -215.4 -428.6 -443.5 -380.2

-142.4 -137.5 -136.7 -146.2 -177.5 -177.9 -126.6

Calciumwater -201.0 -45.0 -41.8 -62.1 -44.9 -8.6 -21.1 -58.5

Proteinwater -2489.8 -2510.1 -2511.3 -3036.8 -2524.2 -2493.7 -2500.0 -2532.0

Waterwater -3507.6 -3169.4 -3070.7 -9117.9 -3 168.9 -3173.1 -3146.0 -3191.3

*Energies are in kcallmol.

TABLE IV. Protein ResiduesLigand Interactions* Resi-

Substrate-A

due'

vdW Cou

Leu-2 Phe-5 Asn-6 Leu-19 Phe-22 Asn-23 Tyr-28 Cys-29 Gly-30 Leu-31 Gly-32 Cys-45 His-48 Asp-49 Tyr-69 Asp-99

-3.8 -2.2 -1.6 -2.2 -2.7 -2.6 -1.6 -2.7 -3.4 -5.9 -0.6 -1.6 -1.5 -2.8 -0.5 0.0

Tnhl

-0.3 0.1 0.0 0.1 -0.1 -0.1 3.8 -1.0 9.3 -5.1 4.8 -7.8 -3.8 43.1 -0.3 -0.1

Substrate-B

Tot -4.0 -2.2 -1.6 -2.1 -2.8 -2.7 2.2 -3.7 5.9 -11.0 4.2 -9.5 -5.3 40.4 -0.8 -0.1

vdW Cou Tot -3.6 -2.2 -1.5 -3.3 -2.7 -3.1 -1.6 -2.7 -3.3 -5.3 -0.7 -1.5 -1.4 -2.8 -2.8 0.0

0.0 0.1 0.0 0.1 -0.2

-3.6 -2.1 -1.5 -3.2 -2.9 -3.3 1.5 -3.8 6.8 -9.5 4.7 -9.3 -5.1 39.5 -8.8 -0.1

Substrate4 vdW

-3.8 -0.2 -3.1 0.1 -0.2 0.1 -3.1 -0.1 -3.2 0.2 -3.3 -0.8 -0.5 -1.5 -1.8 -2.8 -5.5 9.5 -5.8 3.3 -1.7 2.4 -1.4 -3.6 -3.1 -4.2 -3.1 43.9 -2.5 -25.2 -0.1 -0.3

-0.1 3.1 -1.0 10.0 -4.2 5.4 -7.8 -3.7 42.3 -6.0 -0.1 6.9 -31.3 37.9 -0.6 -42.2

-35.7 42.6

Cou

Enantiomer

Tot -4.0 -3.0 -0.1 -3.2 -3.0 -4.1 -2.0 -4.6 4.0 -2.5 0.7 -5.0 -7.3 40.8 -27.7 -0.4

vdW Cou -3.3 -2.3 -1.3 -2.1 -2.8 -3.5 -1.1 -2.1 -4.5 -6.5 0.3 -1.4 -2.0 -2.4 -0.3 0.0

-0.5 -0.3 0.2 0.1 0.3 -0.2 1.9 -0.4 11.4 -5.0 2.7 -7.5 -3.7 43.3 -0.3 0.0

20.8 -21.4 -35.3 42.0

Proton-relay

Charge-separation

Tot

vdW

Cou

Tot

-3.8 -2.6 -1.1 -2.0 -2.4 -3.7 0.9 -2.6 6.9 -11.5 3.0 -8.9 -5.8 40.9 -0.6 0.0

-3.9 -2.9 -1.4 -2.4 -2.9 -2.7 -1.0 -2.4 -1.6 -6.1 -2.1 -2.2 -2.5 -3.5 -0.4 0.0

-6.0 -3.2 0.1 0.1 -4.2 0.8 -6.1 -8.9 7.1 -4.5 5.1 -6.1 -12.6 78.7 -0.1 -0.3

-9.9 -6.1 -1.3 -2.4 -7.1 -1.9 -7.1 -11.3 5.5 -10.6 3.1 -8.3 -15.1 75.2 -0.5 -0.3

6.7 -38.0

39.9

vdW

Cou

-3.2 -6.6 -1.5 -0.8 -1.4 0.0 -2.3 0.1 -2.9 -4.4 -2.2 0.4 -1.3 -1.2 -2.7 -9.6 -0.8 5.6 -4.9 1.0 -2.2 8.5 -1.8 -9.8 -1.4 -77.2 -3.3 96.1 -0.5 -0.6 0.0 1.3

1.9 -32.4

Tot -9.8 -2.3 -1.4 -2.2 -7.3 -1.8 -2.5 -12.3 4.8 -3.9 6.3 -11.7 -78.7 92.8 -1.1 1.3

Inhibitor vdW

Cou

-3.1 -0.5 0.0 -2.2 -1.4 0.0 -3.7 -0.2 -3.4 -5.0 -2.4 0.4 -0.7 -3.5 -1.3 -4.4 -4.1 11.3 -4.2 2.3 -2.5 5.1 -3.0 -6.0 -2.7 -65.0 -3.3 91.9 -0.5 -0.1 0.0 1.0

2.8 -29.8 -38.5

Tot -3.6 -2.2 -1.3 -3.9 -8.4 -2.0 -4.2 -5.7 7.1 -1.9 2.6 -8.9 -67.7 88.7 -0.6 1.0

27.3 -15.2

*Energies are in kcaUmol. 'All protein residues within 3.0 A of a lipid atom or the calcium ion, and the catalytic couple, His-48-Asp-99, are included.

TABLE V. Protein ResiduesCalcium Interactions* Residue'

Substrate-A vdW Cou

Tot

Substrate-B vdW Cou

Tot

Substrate-C vdW Cou

Tot

Enantiomer vdW Cou

Tot

Proton-Relay vdW Cou

Tot

Charge-separation vdW Cou

Tot

Inhibitor vdW Cou

Tot

Phe-5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.1 0.0 0.0 0.0 0.0 1.3 1.3 0.0 0.3 0.3 0.0 0.3 0.3 0.0 0.0 0.0 0.0 0.0 0.0 -0.6 -0.6 0.0 0.0 0.0 0.0 -0.6 -0.6 0.0 0.0 0.0 0.0 0.0 0.0 Phe-22 0.0 Tyr-28 2.9 -27.8 -25.0 2.8 -28.1 -25.3 -0.3 -20.3 -20.6 1.6 -26.4 -24.8 2.9 -30.6 -27.7 2.3 -29.0 -26.7 2.3 -30.6 -28.2 -0.9 -0.1 -0.5 -0.6 -0.2 3.2 3.0 -0.1 -0.8 -0.9 -0.3 4.9 4.7 -0.2 3.6 3.4 -0.3 3.8 3.5 Cys-29 -0.1 -0.8 Gly-30 2.6 -32.1 -29.6 2.4 -31.6 -29.2 0.7 -35.1 -34.4 2.8 -32.1 -29.2 2.8 -34.4 -31.5 2.1 -33.2 -31.0 3.1 -38.4 -35.3 Leu-31 -0.2 2.9 2.7 -0.2 2.0 1.9 -0.1 -0.2 -0.3 -0.2 2.8 2.6 -0.1 -0.3 -0.5 -0.1 -0.2 -0.4 -0.1 -4.0 -4.1 Gly-32 -0.4 -20.4 -20.8 -0.3 -21.1 -21.4 -0.3 -15.9 -16.2 -0.2 -23.6 -23.8 -0.2 -10.5 -10.7 -0.3 -14.4 -14.7 -0.3 -14.2 -14.4 Cys-45 -0.1 2.5 2.4-0.1 2.5 2.5 -0.1 -0.1 -0.2 -0.1 2.8 2.7 -0.1 0.4 0.2 -0.1 3.7 3.7 -0.1 5.9 5.8 3.7 3.7 0.0 -2.9 -2.9 0.0 3.3 3.3 0.0 10.0 10.0 0.0 15.8 15.8 0.0 17.5 17.5 His-48 0.0 4.0 4.0 0.0 Asp49 -0.2 -134.2 -134.4 -0.2 -134.7 -134.9 4.7 -213.1 -208.4 -0.2 -144.2 -144.4 -0.2 -126.7 -126.9 -0.2 -131.5 -131.7 -0.2 -135.2 -135.4 Tyr-69 0.0 0.0 0.0 0.0 1.4 1.4 0.0 6.0 6.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Total

4.5 -205.9 -201.6

4.3 -206.4 -201.9

4.4 -278.9 -274.5

3.7 -218.2 -214.5

4.8 -186.5 -181.7

3.5 -184.9 -181.3

4.4 -194.8 -190.3

*Energies are in kcalimol. 'All protein residues within 3.0 A of the calcium ion, and the catalytic couple, His-48-Asp-99, are included.

complex.)These less favorable interactions are more than compensated by the interactions of the lipid with the protein, calcium, and solvent. The energetics of the substrate complexes A and B are very similar (see Tables I1 and 111)except that the PLA-lipid interaction in complex B is improved due to the better interactions between the lipid and Tyr-69. In complex C the substrateprotein and calcium-pro-

tein interactions are improved significantly, but the ligand-calcium interaction is reduced. Partitioning of the intermolecular energies to residue-residue components gives further insight into the nature of the environment of the substrate and the calcium ion (Tables IV and V). Residues Leu-2, Phe-5, Asn-6, Leu-19, Phe-22, and Asn-23 interact with the sn-2 hydrocarbon chain and are therefore

MODELING OF LIGAND BINDING TO PLA,

van der Waals type interactions. Cys-29, Cys-45, His-48, and Leu-31 are closer to the polar groups of the sn-2 and sn-1 chains and thus have a Coulombic interaction as well as a van der Waals component. Tyr-69 has a negligible interaction with the substrate in complex A but a substantial van der Waals and strong coulomb interaction with the sn-1 carbonyl of the lipid in complex B, and a n even more favorable interaction with the phosphate group (which is charged) in complex C. Four residues in the active site, Tyr-28, Gly-30, Gly-32, and Asp-49, have a nonfavorable interaction (positive) with the substrate. All of these residues, as well as the phosphate group of the substrate, are involved in interactions with the calcium ion (Table IV). Thus, the very strong interactions with the calcium more than compensate for the unfavorable interactions between the similarly charged groups of these protein residues and the substrate. Gly-30 is particularly interesting since its NH group interacts directly with the sn-2 carbonyl and was thus expected to stabilize the substrate. However, this favorable interaction is offset by the strong coulombic repulsion between the carbonyl of this glycine and the substrate’s phosphate group. Hence, although this glycine serves to polarize and stabilize the sn-2 carbonyl group of the lipid, it does not contribute to the overall binding energy.

Dynamic aspects The minimizations of the apoenzyme and of the enzymesubstrate complex were followed by molecular dynamics simulations, in order to investigate the nature and importance of the dynamic behavior of the molecules involved. The details of this study will be presented e l ~ e w h e r eHere . ~ ~ we will focus on the ability of the single structures, as obtained from the minimizations, to represent the major features of complexation in solution. In the molecular dynamics trajectory of the apoenzyme all the typical features of the calcium binding and of the catalytic network were maintained. The calcium ion is coordinated to the carbonyls of Tyr28, Gly-30, and Gly-32, and the carboxylate of Asp49, with average distances (and standard deviations) of 2.39,2.53,3.45, and 2.60 A (0.07, 0.14,0.31, and 0.19 A), respectively. It is also coordinated to 3 water molecules with average distance of 2.39 A and standard deviation of 0.07 A. The catalytic network comprises the His-48-Asp-99 couple, the “active” water which is hydrogen bonded to the His, the two conserved Tyr residues (52 and 731, which are hydrogen bonded to the Asp, and Ala-1, which interacts with the Asp through a water molecule. A 180” rotation around xz of Asp-99 interchanges the positions of the two oxygens, at about 6psec. However, most of the hydrogen bonded network is maintained at all times. The distances (and standard deviations) from His-48 to the active water and to Asp-49 are

55

3.10 and 2.08 A (0.25 and 0.19 A). The average distances Asp-99-Tyr-52, Asp-99-water, and Ala1-water are 1.70,2.70, and 3.08 A, respectively (SD 0.16, 0.20, and 0.16 A). Thus, we have established that the majority of the features observed in the crystal structure which have been suggested to be important for the enzyme’s mechanism were maintained. This validates the adequacy of the energy surface and methodology we used. In the trajectory of the enzyme-substrate complex (56psec) the general geometry as well as most of the details of ligand binding, calcium coordination, and the catalytic network were preserved. The average distances from the ligand’s phosphate to Tyr-69 and the calcium ion, and the distance between the sn-2 carbonyl and the calcium are 1.49, 2.22, and 2.55 A. (SD 0.15, 0.04, and 0.13). However, the average distances from the sn-2 carbonyl to the active water and the NH of Gly-30 have increased to 3.70 and 3.96 A (SD 0.25 and 0.63 A). The active water moved away significantly from His-48 toward the calcium and Asp-49. The rest of the catalytic network was retained, with average distances from Asp-99 to His48, Tyr-52, and Tyr-73 of 2.42 (0.33), 1.61(O.ll), and 1.69 (0.16) A. In addition, after about lOpsec a direct hydrogen bond was formed between the Ala-1 and Asp-99. During the simulation 5 out of the 7 coordinations of the calcium were maintained. In addition to the two interactions with the ligand (above), the average distances to Tyr-28, Gly-32, and a water molecule are 2.39 (0.081, 3.21 (0.17), and 2.40 (0.06) A. Asp-49 moves away slightly and is now coordinated through a water molecule, and the carbonyl of Gly-30 is replaced after about 5psec by the carbonyl of Leu-31. These two residues are part of the surface loop 30-35 which displayed large mobility in a time averaged crystallographically restrained molecular dynamics of bovine PLA,.37 The ligand itself undergoes quite large conformational fluctuations during the simulation, as shown in Figure 3. However, it is important to note that the atoms a t the vicinity of the reaction center and the sn-3 chain have very small fluctuations (SD of ~ 0 . 3 A). The fluctuations get larger at the end of the chains and are significantly bigger for the sn-1 chain than for the sn-2 chain. The standard deviations for the sn-2 atoms Cl-C4, C 5 4 1 0 , and C11 are 0.3, 0.5, and 0.6 A, respectively. The atomic deviations in the sn-1 chain are -0.6, 0.8, 1.1, and 1.5 A, for C 1 4 5 , C 6 4 7 , C8-C9, and C10-C11, respectively. Since the end of the sn-2 chain and most of the sn-1 chain have no interactions with the protein or calcium, the observed fluctuations will not affect the energetics of the complex. In fact, when the enzyme acts with lipid bilayers, the parallel orientation of the two chains will be the more relevant The transient structure after 56psec was minimized and the energetics were compared to those of

56

R.B. SESSIONS ET AL.

SN3 Fig. 3. The mobility of the substrate as revealed in a molecular dynamics simulation of the substrateenzyme complex. Transient structures at intervals of 2 psec along the 56 psec trajectory are shown.

the initial structure. It seems that after a few picoseconds some residues which had repulsive interactions with the ligand (e.g., Gly-30 and Asp-49) were rearranged, and the ligand is docked somewhat deeper in the cleft, resulting in better ligand-protein interactions. Although the active water maintained its position during the simulation of the apoenzyme, it moved from His-48 closer to the calcium in the simulation of the complex. This behavior can be explained by the fact that the active water is poised at the beginning of the simulation very close to the sn-2 carbonyl. This metastable arrangement (or local minimum) is ideal for initiating the reaction. However, since bond formation and dissociation are not possible in molecular mechanics calculations, a more favorable position is found for the water after a few picoseconds of molecular dynamics. Enzyme-Reaction Intermediate Complexes Ester hydrolysis proceeds in two stages via a highenergy tetrahedral intermediate. The rate-limiting

step in the simple acid- or base-catalyzed reaction is known to be the initial addition of the nucleophile to the carbonyl carbon of the ester. It has recently been shown by isotope labeling experiments that this is also likely to be the case PLA, (i.e., the hydrolytic step, or the one before, is rate limiting).39 In the context of the PLA, reaction two scenarios may be envisaged, analogous to the situation that occurs in the serine pro tease^.^'-^' In the “charge separation” mechanism (Diagram 1) the water becomes the active nucleophile by transfer of a proton to NS of His-48. The positively charged His-48 is stabilized by its interaction with Asp-99. The negatively charged, largely desolvated, hydroxide ion attacks the carbonyl carbon of the polarized ester group to produce the tetrahedral intermediate of ester hydrolysis; the negative charge is developed on the old carbonyl oxygen which is coordinated to the calcium ion and hydrogen bonded to the NH of Gly30. Another possible mechanism is the “proton relay” mechanism (Diagram 2). In this case two protons are transferred: the proton from the water molecule

MODELING OF LIGAND BINDING TO PLA,

57

Ca*

0

/An-

+

..... H .N4N . H ..._. ...

w

+

OH

HOPG

Diagram 1.

is transferred to the NS of His-48 and the proton form N' of His-48 is transferred to Asp-99. This is followed by attack of the hydroxide on the carbonyl carbon of the ester as before. This effectively shunts the negative charge originally on Asp-99 first to the water oxygen and then to the old carbonyl oxygen. For the enzyme to perform its catalytic function, we would expect it to stabilize the transition state of the reaction. According to the Hammond postulate43 the transition state is expected to be closer in structure to the unstable tetrahedral intermediate than to the substrate. In which case the tetrahedral intermediate ought to be bound tightly to the protein at the end of the first step of the reaction. Two complexes corresponding to the two possible mechanisms were set up by altering the connectivity of complex A appropriately. These two systems were minimized (initially with harmonic bond potentials and no cross terms until the bond lengths of the new bonds became reasonable). The charge separation system rapidly converged to a local minimum in 1,800 steps while the proton relay system required 5,600 steps to reach the convergence criterion. The rms deviation between the protein of the substrateA complex and the charge separation complex is

0.24 compared with a value of 0.49 for substrate-A and proton relay complexes indicating a greater degree of protein reorganization in the latter case. After fitting the heavy atoms of the protein, the relative deviation of the calcium in the charge separation and proton relay complexes is 0.4 and 1.0 A, respectively. The corresponding deviations of the lipid are 0.63 and 1.0 A, respectively. Inspection of the minimized tetrahedral intermediate structures (Fig. 4) shows that the charge separation complex has retained the general geometry in the region of the reaction center, with a hydrogen bond (2.88 A) between the Ns-H of His-48 and the hydroxyl oxygen of the tetrahedral intermediate. In the proton relay complex the newly formed hydroxyl group of the tetrahedral intermediate is somewhat further from the histidine (3.21 A) and is now in the first coordination sphere of the calcium. In addition, only one of the phosphate oxygens is coordinated to the calcium in the proton relay complex, whereas two are coordinated in the charge separation complex. The differences in structural arrangements are reflected in the corresponding energies associated with the complexes. As seen in Table 11, the intramolecular nonbonded interactions are significantly

R.B. SESSIONS ET AL.

58

U

7

H Caw

0

+

HOPG

OH

ca++

Diagram 2.

more favorable in the charge separation complex (-82 kcallmol). Most of the difference ( ~ 6 kcal/ 0 mol) originates from the stronger electrostatic interactions of the charged His and Asp with the rest of the protein. Another significant difference in intramolecular energy is the higher strain energy of the lipid in the charge separation complex. This is due to the fact that two phosphate oxygens coordinate to the calcium ion and therefore have a small (and strained) 0-P-0 angle (98"). In the proton relay complex only one oxygen is coordinated to the calcium, and the 0-P-0 angle opens up to more tetrahedral geometry (103") which is not as strained. In terms of intermolecular interactions Table I1 shows that the lipid is in a more favorable environment in the charge separation complex whereas all other components of the two complexes have similar energies. A further breakdown of the energetics of the two complexes into molecule-molecule components is given in Table 111. The lipid has better interactions with both the protein and the calcium in the charge separation complex, while the interaction between the calcium and the protein is more favorable in the proton relay complex. The better interactions of the lipid with the protein in the

charge separation complex is mainly due to electrostatic interactions between the negatively charged intermediate and His-48, which is uncharged in the proton relay complex and charged in the charge separation complex (see also Table 11).Both complexes of the intermediate have stronger interactions between the calcium and the lipid, compared with the substrate complex. The better lipid-calcium interactions in the charge separation complex are due to the fact that the calcium interacts with two phosphate oxygens whereas only one of these oxygens is within the first coordination shell of the calcium in the proton relay complex. The coordination of both oxygens of the reaction center to the metal in the proton relay complex reduces this trend, but does not fully compensate for it because of the proximity of the hydroxyl hydrogen which has a partial positive charge. In conclusion, the total energetics of the charge separation complex are more favorable. This energy difference is of the same size as the difference in proton affinities between the imidazole (220 kcal/ mol) and the carboxylic acid (340 kcal/mol) groups (which are not accounted for in the molecular mechanics calculation). However, the larger structural

MODELING OF LIGAND BINDING TO PLA,

59

a

b

C

P

d%

Fig. 4. Stereo picture of the active site of the minimized transition-state enzyme complexes. (a) Charge separation complex; (b) proton relay complex; (c) phosphonate inhibitor-a transition state analogue. Ligand bonds are represented by filled bonds.

Hydrogen bonds are represented by dashed or dotted lines (for d+, 5 2.2 A and 2.2 5 dH...o5 2.6 A, respectively). The calcium ion is indicated by + + and distances to coordinated atoms are marked by dashed and dotted lines. "

"

60

R.B. SESSIONS ET AL.

rearrangement that occurred during the minimization for the proton relay complex suggests that the charge separation model is more plausible. It should also be noted that the proton transfer from the His to Asp resulted in a trans conformation of the acidic group which is not as favorable as the cis conformation. Rotating the corresponding torsion results in a clash with the hydroxyl of Tyr-52, as well as disruption of the stabilizing AspHis hydrogen bond. These results provide support for the charge separation mechanism operating in the PLA,-catalyzed hydrolysis of phospholipids, rather than the proton relay mechanism. This agrees with a recent ab initio study on a model PLA, system44 and also with e ~ p e r i m e n t a land ~~ t h e ~ r e t i c a l ~ ’ . ~ studies ~ - ~ ~ on the serine proteases, which also support the charge separation mechanism for this type of reaction.

Possible Secondary Electrophile In a recent ~ t u d y ~ of ~ ,the ~ ’ X-ray structure of a complex of N . n atra PLA, and a phosphonate inhibitor, a second calcium ion was observed in the vicinity of the ligand, coordinated to the carbonyl of residue 29. It was proposed that this second calcium is of importance for the function of the enzyme and serves as an additional electrophile which stabilizes the transition state anion. There are no other studies suggesting the existence or importance of a second calcium, and it was not observed in the crystal of the bee venom ~omplex.~’ We have investigated the possibility of incorporating a second electrophile in our models of substrate and transition state complexes and its effect on the energetics of the system. We have replaced the water molecule that is hydrogen bonded to the carbonyl of Cys-29 with a calcium ion in the complex with the substrate (C) and in the complex with the charge separation tetrahedral intermediate and reminirni~ed.~’ In both complexes the calcium ion was accommodated without disruption of the general structure. The rms deviation between the backbone atoms of the substrate complexes with and without secondary calcium was 0.55 A. The corresponding rms for the tetrahedral intermediate complex was 0.48 A. In both complexes the coordination of the first calcium and the interactions of the ligand were retained. In the substrate complex the secondary calcium is coordinated to the carbonyls of residues 25-29 and 31. In the tetrahedral intermediate complex this calcium is coordinated only to four carbonyls 25 and 27-29 and to two water molecules. Tyr-28 coordinates both calciums in these complexes. Energy analysis of the two complexes reveals that inclusion of a secondary calcium introduces a large repulsion between the two cations, as expected, but this is more than compensated by stabilizing other intermolecular interactions. The overall stabilization is larger in the tetrahedral intermediate complex than in the substrate complex. The main source

of excess stabilization of the tetrahedral intermediate is the interaction between the secondary calcium and the ligand which carries an extra negative charge in the intermediate. Additional stabilization of the transition state would occur if the secondary calcium polarizes the amide bond between residues 29 and 30.This effect cannot be investigated by using the current level of molecular mechanics calculations since they use permanent atomic partial charges. The importance of this effect could be studied only by using small model systems and employing quantum mechanics calculations or molecular mechanics calculations which incorporate polarizabilities. Modified Substrates and Inhibitors In order to further investigate the validity of our proposed model for ligand binding to the enzyme, we examined its ability to account for the protein’s stereospecificity and behavior toward modified substrates and inhibitors.

Stereospecificity PLA, is stereospecific and hydrolyzes only the phospholipid with the correct stereochemistry (L). Although the D enantiomer cannot be hydrolyzed, it binds to the enzyme just as strongly and acts as a competitive inhibitor. We have generated the mirror image of the phospholipid in the substrateenzyme complex, and by adjusting the torsions of the sn-3 chain managed to dock this enantiomer into the active site. This new complex was minimized and the active site region of the minimized complex is shown in Figure 2d. As can be seen by comparing Figures 2a and 3d the general position of the three chains is similar, and the phosphate group is coordinated to the calcium ion. The total energy and the various components of the two complexes are very similar, in agreement with their similar experimental binding constants. However, for the “wrong” (D) enantiomer the sn-2 carbonyl is pointing in a different direction and thus is not polarized by the calcium ion and the NH of Gly-30, and is not located in a suitable position for an attack by the “active water.” It has been shown experimentally3’ that the Tyr69 residue is important for stereospecificity. While the native enzyme hydrolyzes only the L enantiomer a Phe-69 mutant hydrolyzes both (although the D enantiomer a t a much reduced rate). In addition, whereas the native enzyme only hydrolyzes the R, sulfur substrate, the Phe-69 mutant hydrolyzes both the R, and S, sulfur analogues. Thus, our model (C) explains a t the atomic level the stereospecificity of the enzyme: the Tyr-69 fixes the position and orientation of the phosphate of the substrate so that both calcium binding and correct reaction center orientation can occur only for the L enantiomer and for the R,sulfur analogue. When Tyr is replaced by Phe the

MODELING OF LIGAND BINDING TO PLA,

61

Fig. 5 . The 1-R all-trans cyclopentane-l,2,3-triol (filled bonds) after constrained minimization, superimposed on to the minimized receptor site conformation of the substrate.

orientation of the phosphate can be changed to accommodate the “wrong” enantiomers.

Conformatiomlly restricted ligands Since the model substrate is quite flexible and could, i n principle, adopt many conformations it is useful to examine analogues with restricted conformational freedom in order to test the plausibility of the docked lipid in our model. We examined two types of analogues-a tetrahydrofuranone (11)and a cyclopentano-lipid (111).The first one is a n inhibitor in which the sn-1 and sn-2 chains are linked by a 5-membered lactone ring, and the other is a substrate in which the sn-1 and sn-3 positions are linked by a n ethylene group to form a cyclopentane ring. The lipid 3-arachidonyl-4(0-phosphoethanolamino)-methyletrahydrofuran-2-onewas found to be a potent inhibitor of PLA,.16 This molecule could adopt cis or trans conformations or exist as E or Z exocyclic enols, resulting in eight distinct chiral isomers. Each one of these structures was built and their conformations were adjusted to mimic the docked substrate by constrained minimisations. A good fit to the model substrate was obtained for the (3S,4R) structure. In this structure the sn-2 carbonyl (and the rest of this chain), and the sn-3 chain overlap the corresponding groups of the model substrate, and the lactone ring is in the plane defined by the sn-1 and sn-2 chains of the substrate. All other structures had large deviations from the model substrate or a n unfavorable orientation of the lactone ring. A set of conformationally restricted phospholipid analogues based on 1,2,3-cyclopentanetriol has been synthesized and its susceptibility toward hydrolysis by both venom and pancreatic PLA,s investi-

It was observed that of the six possible positional/stereoisomers only four showed qualitative evidence of hydrolysis by PLA,. Of this subset, only one enantiomer (laevorotatory) of the all-trans (1P) isomer showed a measurable rate of reaction in the presence of PLA,. Figure 5 shows how the 1-R enantiomer of this isomer can overlay the substrate model in the proposed active site conformation. The cyclopentane ring reduces the number of available conformations of the substrate, and in particular restricts the relative positions of the phosphate and the two carbonyl groups. A pucker of the cyclopentane ring such that all ring substituents are pseudoequatorial allows the overlay of the sn-1 and sn-2 carbonyls with those of the lipid in the complex. The position of the phosphate in relation to these groups is determined by the Cl-O(P) torsion. In the original lipid this torsion angle can adopt three possible staggered conformations, whereas the cyclopentane analogue has only limited freedom determined by the ring. The pucker that is required to get the appropriate relative orientation of the two carbonyls results in positioning the phosphate in a staggered conformation corresponding to the one in the minimized complex. The fact that the phosphate of the substrate model occupies the intersection of the large conformational space available to the lipid with the restricted space available to the cyclopentano-lipid strongly supports the validity of the model. It is clear from Figure 5 that altering the configuration a t one or more of the chiral centers of the cyclopentane-phospholipid would prevent the successful overlay of the analogue with the docked substrate. On the other hand, in the absence of the constraints provided by the proposed model, it is perfectly possible to overlay either the (R)- or (S)-

62

R.B. SESSIONS ET AL.

enantiomer of a phospholipid onto any one of the possible cis-trans or all-cis isomers of the (1P)-cyclopentane-phospholipid by adjusting appropriate phospholipid torsions. Hence the proposed model is consistent with the experimental results and allows the prediction that the active (-)-enantiomer will have 1-R absolute configuration.

Conformationally bulky substrates A series of phospholipids with alkyl branches on the fatty acid side chains have been synthesized and the structure activity relationship of their hydrolysis by snake venom PLA, was determined.56 Substituents were introduced into just the sn-2 chain or into both the sn-1 and the sn-2 chains. The protein is fairly tolerant of a methyl introduced between positions 4 to 8. This tolerance rapidly diminishes as the size of the alkyl substituent is increased. However, substitutions at position 12 of groups as large as butyl or phenyl have little or no effect upon reactivity. If the substrate model is examined it can be seen that the two acyl side chains protrude out of the active site, the sn-2 chain breaks the surface around carbon 10-11, and the sn-1 around carbon 5-6. Thus substitution a t position 12 of the sn-2 chain will not cause any clashes with the enzyme. The ability to introduce small substituents onto the lipid a t positions inside the active site is consistent with the large and rather flexible” nature of the cavity. In fact there are a few water molecules in the active site (see Fig. 2a) which could be eliminated when substituted lipids are docked. Transition state surrogate inhibitor A number of transition state surrogate inhibitors of PLA, have been prepared which mimic the geometry and charge of the tetrahedral intermediate of substrate h y d r o l y s i ~ .The ~ ~ most . ~ ~ potent of these is (IV). The binding of IV to PLA, was modeled by replacing the tetrahedral intermediate in the charge separation complex with IV in a similar conformation. The minimized complex retains the general features of the charge separation complex, with rms deviations of 0.46 and 0.61 A for backbone and all heavy atoms, respectively. The similarity in structure in the active site region can be seen by comparing Figure 4b and c. A hydrogen bond exists between an oxygen of the “sn-2” phosphonate and N8 of His48. The other phosphonate oxygen is hydrogen bonded to the NH of Gly-30 and coordinated to the calcium ion. The phosphate group of the ligand is also coordinated to the calcium ion. The energetics of the complex are summarized in Table 11, the molecule-molecule interactions are given in Table 111, and the ligand and calcium interactions with neighbowing residues in Tables IV and V. As discussed above the tetrahedral intermediate forms a more stable complex with the enzyme than the substrate. In general the total energies and the various molec-

ular and residue components of the energy are more favorable for the inhibitor than for the substrate, but somewhat less favorable than those of the corresponding tetrahedral intermediate complex. This is mainly due to the similarity in charge distribution between the inhibitor and the reaction intermediate. This demonstrates the underlying reason for the efficacy of transition state analogues as inhibitors. By mimicking the transition state (or the very similar intermediate) in terms of internal structure and charge distribution, it is possible to achieve a tighter binding than by mimicking the substrate’s structure and charges. An X-ray structure of the complex of this inhibitor with cobra49 and bee5’ venom PLA, was published recently and verified all the important features of our model.

CONCLUSIONS A model of the complex between PLA, and a substrate was developed by docking a phospholipid in the active site cavity of the enzyme, followed by an energy minimization. This was achieved with minor modifications to the structure of the lipid as observed in crystalline state and in solution. Some adjustments to the position of a few residues a t the entrance to the cavity were required in order to achieve optimal interactions with the protein and the calcium ion. The final complex (Fig. 2c) has all the features consistent with the suggested mechanism: The residue His-48 is hydrogen bonded to Asp99 (forming the catalytic couple), and to a water 3 from the carbonyl molecule which is poised ~ 3 . A carbon of the reaction center. This carbonyl is polarked by the NH of Gly-30 and by the calcium ion. The phosphate group is coordinated to the calcium ion and to the OH group of the side chain of Tyr-69. Based on this complex the stereoselectivity of the native enzyme and of a Phe-69 mutant toward substrates could be explained at the atomic level. Examples of a substrate and an inhibitor with conformational restrictions were used to further validate the model. Two alternative models for the reaction intermediate complex were examined, which correspond to the suggested charge separation and the proton transfer mechanisms. The structural and energetic differences between the two complexes supported the charge separation mechanism as more plausible. Analysis of the minimized complex of the enzyme with the phosphonate inhibitor (IV) revealed substantial similarities to the corresponding complex of the tetrahedral intermediate, in terms of structure and energetics, accounting for the efficacy of transition state analogues as inhibitors.

ACKNOWLEDGMENTS We would like to acknowledge the use of the SERC funded Chemical Databank services at Daresbury for part of the work described in this paper. The

MODELING OF LIGAND BINDING TO PLA,

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57.

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