Biochimie (1992) 74, 859-866 © Soci6t6 franfaise de biochimie et biologie mol6culaire / Elsevier, Pads

859

NMR studies of interactions between inhibitors and porcine pancreatic phospholipase A 2 AR Peters 1, N Dekker~,2, L van den Berg 2, R Boelens l, AJ Slotboom 2, GH de Haas 2, R Kaptein~* tBijvoet Center, Department of Chemistry; 2Laboratory of Biochemistry, CBLE, State University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands (Received 27 May 1992; accepted 3 August 1992)

Summary - - Two-dimensional NMR studies were performed on the complexes of porcine pancreatic phospholipase A2, bound to a micellar lipid-water interface of fully deuterated dodecylphosphocholine, with competitive inhibitors derived from the following general structure: C-X H I Y-C-N-C-H II I O C-OPO3C2H4OH X and Y are alkyl chains with various 'reporter groups'. The interactions between the inhibitor and the enzyme were localized by comparison of 2-D nuclear Overhauser effect spectra using protonated and selectively deuterated inhibitors, and inhibitors with groups having easily identifiable chemical shifts. These experiments led us to the following conclusions for the phospholipase A2/inhibitor/micelle complex: i) the His48 C2 ring proton is in close proximity to both the amide proton and the methylene protons at the sn-I position of the glycerol skeleton of the inhibitor; ii) the acyl chain of the inhibitor at the sn-2 position makes hydrophobic contacts near Phe5, lie9, Phe22 and Phel06; iii) no interactions between the acyl chain at the sn-I position and the protein could be identified. Comparison of our results on the enzyme/inhibitor/micelle ternary complex with the crystal structure of the enzymeinhibitor complex shows that the mode cf inhibitor binding is similar. However, in several cases we found indications that the hydrophobic chains of the inhibitors can have multiple conformations. phospholipase A 2 / competitive inhibitors / protein.inhibitor interactions / micellar interface

Introduction

Currently there is a great interest in the design of inhibitors to phospholipase A2. Synthetic competitive inhibitors having a 2-amide instead of a 2-ester linkage were shown to be powerful inhibitors of porcine pancreatic phospholipase A2 (PLA), but only when the enzyme is bound to an interface [ 1]. The crystal structure of a complex of a porcine pancreatic PLA mutant and a competitive inhibitor of this type has been reported [2]. Substrate-like inhibitors wi~h a phosphonate group at the position of the 2-ester linkage have also Iseen shown to be effective, and crystal structures of their complexes (with different PLAs) are available [3-5]. However, the crystallization of protein-

*Correspondence and reprints

inhibitor complexes bound to micelles is difficult or even impossible, and therefore X-ray crystallography cannot give direct information on inhibitor binding under these conditions. Our aim is to use high resolution NMR spectroscopy to obtain detailed information about proteininhibitor complexes bound to a micellar system. The assignment of the NMR spectrum of PLA has been previously reported [6], and a study of a ternary PLA/inhibitor/micelle complex has been made [7]. Here we report the results on specific inter-molecular contacts between inhibitors and PLA, which can be vbtained from intermolecular nuclear Overhauser effects (NOE). Various inhibitors were designed to enable assignment of the NOEs between an inhibitor and PLA. Thus, in a number of inhibitors the protonated acyl-chains were replaced by their deuterated analogues, which allowed us to identify intermolecular NOEs. The assignment of protein reso-

860 nances can usually be made by established methods. However, the specific assignment of the resonances to protons in the acyl-chain is very difficult. For this reason at certain positions in the acyl chains characteristic groups were introduced, which have chemical shifts that can be easily identified.

The data were processed on a DEC Vaxstation with the 'Triton" software package (written in Fortran 77). Each data set was processed with a sine-bell window shifted over lt/6 in oh and over n/4 in tom and zerofilled to obtain a spectral data matrix of I K x I K. After complete Fourier transformation of the spectrum, automatic baseline corrections were applied in both dimensions [ 15,16].

Materials and methods

Results

inhibitors

The inhibitory power Z of the inhibitors has been discussed by Ransac et al [91. In a first approximation, it is equal to the ratio of the Michaelis-Menten constant of the enzyme at a substrate interface, and the dissociation constant of the enzyme-inhibitor complex at the interface. Z-values were determined using the substrate 1.2-didodecanoyl-sn-glycero-3-phosphocholine, as described [10l. For inhibitor 13, the Z-value is referred to the substrate ! ,2-di-(5-phenyi)pentanoyl-sn-glycero-3-phosphocholine, which was used to verify if a lipid with bulky side chains is a substrate tbr PLA. This substrate was found to be hydrolyzed efficiently by the enzyme.

In figure 1 the schematic structures, the abbreviated names, and the Z-values are given for the inhibitors used in this study. In addition, for inhibitor DII the octyl chain from I 1 has been replaced by a deuterated one, and similarly in inhibitor DI2 the acyl chain at the sn-2 position of I2 has been deuterated. This allowed the identification of inhibitor-protein NOEs. As characteristic groups with unique chemical shifts we used double bonds (14-16) or an aromatic ring (13). The inhibitory power Z, which is proportional to the ratio of the affinity of the enzyme for the inhibitor and the substrate, is also given in figure 1. All compounds are good inhibitors as indicated by these Z-values. Since many of the inhibitors are derived from the amino-acid norleucine, in this study atoms are named as for amino acids, and this nomenclature we use in all our inhibitors for easy comparison. Specifically, we refer to the carbon at the position corresponding to the sn-2 position in the glycerol moiety, as Cot. The carbon at the sn-! position we designate C[I, and those in the alkyl chain attached to it as C¥-C¢. The carbon at the sn-3 position is referred to as CI3'. Carbons in the substituent chains at the sn-I or sn-2 position are numbered as C 1 (etc) within that chain counting from the glycerol moiety outwards.

Sample preparation

Observation and assignment of inter-molecular NOEs

The synthesis of l-octyl-2-N-dodecanoylamino-2-deoxy-snglycero-3-phosphoglycol (inhibitor il) and similar lipids has been described [81. The deuterated molecule was synthesised similarly, using fully deuterated octanol. (R)-2-dodecanoylamino-hexanol-l-phosphoglycol (inhibitor 12) and similar lipids were described [1]. The corresponding deuterated inhibitor was synthesized similarly, using fully deuterated lauric acid [7]. The fully deuterated octanol, dodecanoic acid, NaBD4 and CD3I were from Cambridge Isotope Laboratory.

Dodecylphosphocholine (DPC) Fully deuterated dodecylphosphocholine was obtained from Cambridge Isotope Laboratory.

Competitive inhibition at interfaces

The NMR samples were typically 1 mM enzyme in 99.96% 2H:O containing 50 mM CaC! 2. 150 mM NaCi, 100 mM DPC, and 1.2-1.6 mM inhibitor. The pH (uncorrected meter reading) was adjusted to 5.0 by the addition of microliter quantities of dilute solutions of 2HC! and NaO2H. Temperature was set to 313K.

The binding of the inhibitors I I - I 6 to the complex of PLA (1 raM) and DPC-micelles (100 m M ) was studied by 2-D N O E spectroscopy. Relevant NOEs are listed in table I. Since we obtained most results for I2, this inhibitor will be discussed first.

NMR spectroscopy All 2-D NMR spectra were recorded on Bruker AM 600 or Bruker AM 500 spectrometers (the former at the SON hf-NMR facility, Department of Biophysical Chemistry, Nijmegen University, The Netherlands). The 1H2HO signal was suppressed by presaturation during the l-s recycle delay and during the mixing time of the 2-D NOE experiments. The 600 MHz 2-D NOE spectra were recorded with a 32-step phase cycle (cf [1 l l) and a mixing time of 150 ms, and consist of 320 x 2K to 450 x 2K data points. The homonuclear HartmannHahn transfer (HOHAHA) spectra were recorded with MLEV17 [121, clean-TOCSY [131 and DIPSI-2 [141 sequences at 500 MHz with mixing times of 25-35 ms.

lnhihitor 12 The PLA/I2/micelle complex has been discussed before [7]. Part o f the 2-D N O E spectrum is shown in figure 2. In this spectrum a number o f new NOEs could be detected, as compared to the 2-D N O E spectrum of the PLA/micelle complex without inhibitor. A 2-D N O E spectrum was also recorded for the complex using the inhibitor with a deuterated acyl chain at the sn-2 position (DI2). Around 1.3 ppm, which is the chemical shift o f the methylene protons of the inhibitor acyl chain, five N O E s are present to the

861 aromatic region of the spectrum of the complex with the protonated inhibitor. These NOEs are absent in the spectrum of the complex with the deuterated inhibitor, and are therefore intermolecular. The aromatic resonances could be assigned to PheS, Phe22 and Phe 106 on the basis of interresidual NOEs to other aromatic residues and to the aliphatic side chains of Ile9, Leu41 and Alal02. These assignments were confirmed by a 2-D H O H A H A spectrum. In addition, both in the I2- and DI2-PLA complexes there are NOEs from the His48 C2 ring proton (at 6.26 ppm) to several broad resonances at 2.3 to 1.3 ppm. These NOEs could be assigned to intermolecular interactions after comparison with the spectra of I 1 (vide infra). In the high field part of the 2-D NOE spectrum, an NOE cross-peak can be observed from the aliphatic methylene protons of the inhibitor to Ile9. The NOE of the ~ methyl of Ile9 is present in the spectrum of the protonated inhibitor, but is clearly absent in the spectrum of the deuterated inhibitor, and must therefore be intermolecular. Besides the NOE to ca 1.30 ppm, which is the position of the bulk of the alkyl chain, there are NOEs from Ile9 ~i methyl to 1.21 and 1.10 ppm, which are also absent in the spectrum of the deuterated inhibitor.

Inhibitor I1 For I1 the same NOEs could be observed as for I2 (it has the same chain at the sn-2 position), but here resonances from Phe22 and Phel06 are overlapping with each other and with resonances from Tyr75 and Tyrl 1 I. At Ile9, only the NOE to the bulk of the alkyl chain (at 1.29 ppm) is visible. From H O H A H A and NOE spectra recorded in H20, resonances could be assigned to the amide proton of the inhibitor (at 11.50 and 10.55 ppm), and to the inhibitor Cct proton (at 5.07 ppm). The amide proton has an NOE to the His48 C2 ring proton, and it is therefore likely that it is hydrogen-bonded to the N l of the histidine ring, as observed in the crystal structure [2]. For the Ct~ proton, only intra-molecular NOEs could be identified. A comparison of the complex of the inhibitor with the deuterated sn-I chain (DII), with that of the protonated inhibitor, revealed few differences, and only an intramolecular NOE was identified. However, comparison of I1 with 12 yielded indirect identifications of inter-molecular NOEs. The His48 C2 ring proton (at 6.27 ppm) has NOEs to protons at 3.98 (weak), 3.57, and 3.49 (with a tail ca 3.45) ppm. These new resonances are assigned to the methylene protons of the C~ in I1, which is based on the follow-

H2Ca----C~H3

I-I-z.C0)

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H,O,_o/ O I-I,~LoPo3C2H4OH

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Fig 1. Schematic structures of the inhibitors used in this study. Abbreviated names and inhibitory power Z are indicated.

862 Table I. Intermolecular NOEs. a~ (ppm)

Assignment

tol (ppm)

Assignment

Comments

11.50 10.55 7.00 6.82 6.68 6.27 6.27 6.27 6.27 6.27 - 0.02 6.99 6.86 6.~9 6.80 ca 6.72 ca 6.72 6. i !-6.12 6.26 6.26 6.26 0.00 0.00 0.00 0.12 0.02 6.95 6.87 6.87 6.77 6.77 6.70 6.70 6.06 6.0b - 0.01

I 1 NH I 1 NH Phe5(3,5) Phe 106(4), Phe22(2,6) Phe 106(3,5) His48 C2H His48 C2H His48 C2H His48 C2H His48 C2H lie9 5methyl Phe5(3,5) Phe 106(4) Phe22(2,6) Phe22(2,6) Phe106(3,5) Phe I06(3,5) Phe5(4) His48 C2H His48 C2H His48 C2H Ile9 ~methyl Ile9 5methyl lie 9 8methyl lie9 8methyl lie9 ~imethyl Phe5(3,5) Phe 106(4) Phe 106(4) Phe22(2,6) Phe22(2,6) Phe 106(3,5) Phe106(3,5) Phe5(4) Phe5(4) lie9 8methyl Ile9 8methyl Phe5(3,5) or Phel06(4) Phe5(4) His48 C2H His48 C2H

5.07 6.26 1.31-1.33 1.30 1.30 3.97-3.98 3.57 ca 3.49 3.45 5.04-5.07 1.29 1.31 1.31 1.32 1.28-1.29 1.31 1.28-1.30 1.31 2.26 1.63

I 1 C~tH His48 C2H l I sn-2 acyl I 1 sn-2 acyl I l sn-2 acyl

Also in HOHAHA Major conformat

-0.01

6.96 6.19 6.29 6.29

1.44-1.46

ing considerations. Figure 3 compares the crosssections in the 2-D NOE spectra of the complexes of I 1 and 12 at the position of the His48 C2 ring proton. The NOEs visible here are also present in the spectra of the deuterated compounds, and can therefore not be due to protons in the acyl chains. Since the only difference between the samples is in the substitution at the sn-I position of the glycerol skeleton of the inhibitor, the NOEs must be due to protons in that part. From textbook tables a chemical shift of about 3.65 can be expected for the methylene protons at the sn-1 and sn-3 positions in I 1. Note that a resonance at

1.30 1.21 1.14 6.99 5.06 1.34 1.31 I. 18 1.28 1.16 1.30 !. 19 1.28 I. ! 8 1.23 I. 18 2.40 2.40 3.56 3.49

I1 CI3HL2 I1 Call I1 sn-2 acyl I2 sn-2 acyl 12 sn-2 acyl 12 sn-2 acyl I2 sn-2 acyl 12 sn-2 acyi I2 sn-2 acyl I2 sn-2 acyl 12 sn-I alkyl 12 sn-1 alkyl 12 sn- 1 alkyl 12 sn-2 acyl 12 sn-2 acyl 12 sn-2 acyl 13 aromate 14 H (9) or H(l°~ I5 sn-2 acyl 15 sn-2 acyl 15 sn-2 acyl 15 sn-2 acyl 15 sn-2 acyl I5 sn-2 acyl I5 sn-2 acyl I5 sn-2 acyl I5 sn-2 acyl 15 sn-2 acyl I5 sn-2 acyl 16 sn-2Ct2~ orCt3~ 16 sn-2C{2~ orC(3J 16 sn-2C t2) orCt3~ I6 sn-2C t2) orC(3~

Not in 12 Not in 12 Not in 12 Not in 12 Veryweak Not in DI2 Not in DI2 Not in DI2 Not in DI2 Not in DI2 Not in DI2 Not in DI2 Not in II Not in I 1 Not in I 1 Not in DI2 Not in DI2 Not in DI2 Weak

Strong Medium Noisy, weak Noisy, weak

3.57 ppm gives NOEs both to the His48 C2H, and to the sn-2 (Cot) proton at 5.05 ppm. For I2, NOEs are observed from His48 C2H to resonances around 2 ppm (see fig 3, lower trace). This corresponds to resonance positions expected for the 13, ), and further protons in the acyl chain of the norLeu part. NOEs to the methylene protons at the sn-3 (1~') position, which are expected around 3.57 ppm, are absent for I2. All this is consistent with His48 C2 ring proton having an interaction with the sn-I (C13) methylene protons of the glycerol part of the inhibitor. There remains the problem however, that for two methylene

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NMR studies of interactions between inhibitors and porcine pancreatic phospholipase A2.

Two-dimensional NMR studies were performed on the complexes of porcine pancreatic phospholipase A2, bound to a micellar lipid-water interface of fully...
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