Báo cáo Y học: Binding of Thermomyces (Humicola) lanuginosa lipase to the mixed micelles of cis-parinaric acid/NaTDC Fluorescence resonance energy transfer and crystallographic study

Eur. J. Biochem. 269, 1613–1621 (2002) Ó FEBS 2002 Binding of Thermomyces (Humicola) lanuginosa lipase to the mixed micelles of cis-parinaric acid/NaTDC Fluorescence resonance energy transfer and crystallographic study Stephane Yapoudjian1, Margarita G. Ivanova1, A. Marek Brzozowski2, Shamkant A. Patkar3, Jesper Vind3, Allan Svendsen3 and Robert Verger1 1Laboratoire de Lipolyse Enzymatique CNRS-IFR1, Marseille, France; 2Structural Biology Laboratory, Chemistry Department, University of York, UK; 3Enzyme Research, Novozymes A/S, Bagsvaerd, Denmark The binding of Thermomyces lanuginosa lipase and its mutants [TLL(S146A), TLL(W89L), TLL(W117F, W221H,W260H)]tothemixedmicellesofcis-parinaricacid/ sodiumtaurodeoxycholateatpH 5.0ledtothequenchingof theintrinsictryptophanfluorescenceemission(300–380 nm) and to a simultaneous increase in the cis-parinaric acid fluorescence emission (380–500 nm). These findings were used to characterize the Thermomyces lanuginosa lipase/cis-parinaric acid interactions occurring in the presence of sodium taurodeoxycholate.The fluorescence resonance energy transfer and Stern–Volmer quenching constant values obtained were correlated with the accessibility of the tryptophanresiduestothecis-parinaricacidandwiththelid opening ability of Thermomyces lanuginosa lipase (and its mutants). TLL(S146A) was found to have the highest Lipases(EC 3.1.1.3)canbedefinedasenzymesthatcatalyze thehydrolysisoflong-chainacyl-glycerols [1].Inadditionto playing an important role in fat catabolism, they have numerousapplicationsinthefood,cosmetics,detergentand pharmaceutical industries [2–5]. Inrecentyears,thethree-dimensionalstructuresoflipases and lipase–inhibitor complexes have been determined using X-ray crystallographic methods [6–11]. All lipases show a common a–b hydrolase fold [12] and a catalytic triad composed of a nucleophilic serine, which is activated via Correspondence to R. Verger, Laboratoire de Lipolyse Enzymatique, CNRS-IFR1, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France. Fax: + 33 91 71 58 57, Tel.: + 33 91 16 40 93, E-mail: verger@ibsm.cnrs-mrs.fr Abbreviations: cis-PnA, cis-parinaric acid; CMC, critical micellar concentration; FRET, fluorescence resonance energy transfer; KSV, Stern–Volmer quenching constant; NaTDC, sodium taurodeoxycho-late; OA, oleic acid; POPG, 1-palmitoyl-2-oleoylglycero-sn-3-phos-phoglycerol; RFI, relative fluorescence intensity; TLL, Thermomyces lanuginosa lipase; TLL(S146A), inactive mutant with S146 mutated to A; TLL(W89L), mutant with W89 mutated to L; TLL(W117F, W221H, W260H), mutant with only the W89; W117 mutated to F, W221 mutated to H and W260 mutated to H. Note: the atomic co-ordinates have been deposited in the Brookhaven Protein Data Bank with the accession code 1gt6. (Received 20 August 2001, revised 5 December 2001, accepted 14 January 2002) fluorescence resonance energy transfer. In addition, a TLL(S146A)/oleic acid complex was crystallised and its three-dimensional structure was solved. Surprisingly, two possiblebindingmodes(sn-1andantisn1)werefoundtoexist between oleic acid and the catalytic cleft of the open con-formationofTLL(S146A).Bothbindingmodesinvolvedan interactionwithtryptophan89ofthelipaselid,inagreement with fluorescence resonance energy transfer experiments.As a consequence, we concluded that TLL(S146A) mutant is notanappropriatesubstituteforthewild-typeThermomyces lanuginosa lipase for mimicking the interaction between the wild-type enzyme and lipids. Keywords: lipase; X-ray crystallography; cis-parinaric acid; fluorescence resonance energy transfer. hydrogen bonds as partofachargerelaysystem, along with the histidine and the aspartate or glutamate residues [6,7]. The crystal structures of some lipases have shown that the active site is covered by a helical surface loop or ÔlidÕ that renders the active site inaccessible to substrate. This is referred to as the closed conformation of the lipase. On the other hand, the three-dimensional structures of lipases complexedwith inhibitors shows a rearrangementofthelid, allowing free access to the active site in the so-called open conformation, in which a large hydrophobic surface around the catalytic triad is exposed. Thermomyces lanuginosa lipase (TLL) has four trypto-phan residues located in positions 89, 117, 221 and 260. The side chains of W117, W221 and W260 are buried into the protein core, whereas the W89 residue is located in the central part of the helical lid [13]. In the crystal structures of the open forms of TLL, W89 is in close van der Waals contact with the acyl moiety of an inhibitor mimicking the transition state [14,15]. The fluorescence technique was used previously to study the binding of TLL to small or large unilamellar vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG) and to vesicles of zwitterionic phospholipids such as 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine [16]. The authorsconcludedthatTLLmaybindwithasimilaraffinity to all types of phospholipid vesicles and may adopt a catalytically active conformation and be involved in inter-facial activation processes only when small unilamellar vesicles of POPG are used. Furthermore, molecular 1614 S. Yapoudjian et al. (Eur. J. Biochem. 269) dynamics simulations [17] indicated that the replacement of a single amino acid at the active site (S146A) may lead to conformational alterations in TLL. The aim of the present study was to investigate the TLL/ fatty acid interactions using the fluorescence resonance energy transfer (FRET) technique. One of the prerequisites to be able to observe the FRET between a donor (TLL tryptophans) and an acceptor (fatty acid) is that there must exist a spectral overlap between the donor emission and the acceptor absorption spectra, and the donor and acceptor groups must be the right distance apart and properly oriented [18]. Therefore 9,11,13,15-cis,trans,trans,cis-octadecatetraenoic acid (cis-PnA), a naturally fluorescent fatty acid with thoroughly characterized spectroscopic properties [19], was chosen for use as a probe. It has been previously established that cis-PnA can act as an acceptor for the tryptophan fluorescence emission [20], and its spectroscop-ic properties have been used in studies on fatty acid binding to various proteins [20,21]. Bile salts are the main detergent-like molecules respon-sible for the solubilization of lipolytic products (monoglyc-erides and free fatty acids) during the digestion of dietary fats. Sodium taurodeoxycholate (NaTDC) is a conjugated bile acid, which forms very small micelles in an aqueous solution[22].Themixedmicellesofcis-PnA/NaTDCturned out to be a convenient model system for studying the interactions between a water soluble protein (TLL) and a fatty acid in the form of mixed micelles. First, we studied the lipase free cis-PnA/NaTDC system in order to characterize the cis-PnA/NaTDC mixed micellar system. The binding behavior of TLL (and its mutants) to purecis-PnAandtomixedmicellesofcis-PnA/NaTDCwas then studied using the FRET technique. In addition, X-ray crystallogaphy studies were performed on the S146A mutant in order to elucidate the particular properties of its complexes with fatty acids. MATERIALS AND METHODS Materials NaTDC was from Sigma and cis-PnA was from Molecu-lar Probes. A stock solution of 3.2 mM of cis-PnA in ethanol containing 0.001% (w/v) butylhydroxytoluene (BHT) as an antioxydant was stored in the dark at )20 °C under an argon atmosphere. These precautions were taken to ensure that no polyene decomposition would occur [20]. The TLL wild-type, its single mutants: TLL(S146A), TLL(W89L), and triple mutant TLL(W117F, W221H, W260H) were used. All enzymes were kindly provided by A. Svendsen and S. A. Patkar from Novo Nordisk, Denmark and prepared as described previously [23,24]. The buffers used were 10 mM Tris/HCl pH 8.0, 150 mM NaCl, 21 mM CaCl2, 1 mM EDTA and 10 mM acetate pH 5.0, 100 mM NaCl, 20 mM CaCl2. UV absorption spectroscopy Differential absorption spectra were recorded on a Uvikon 860 spectrophotometer from Kontron Instruments. All assays were carried out using two quartz cuvettes (optical Ó FEBS 2002 pathlength1 cm)of3.5 mLeach:onefortheassayandone for the control assay. The contents of each cuvette were mixed 5–10 times by gentle inversion of the cuvette capped with Teflon stopper, and were then left unstirred during the measurement procedure. Measurements were performed at room temperature. Two types of experiments were per-formed. (a) Titration of cis-PnA was carried out by the increasing amounts of NaTDC at pH 5.0, in the absence of TLL. The assay and control cuvettes were both filled with bufferandNaTDCatthevariousconcentrationstested.cis-PnA was subsequently added to the assay cuvette from an ethanolic stock solution and differential absorption spectra were recorded between 200 and 450 mn. (b) Absorption spectra of cis-PnA in the presence of TLL at pH 5.0 or pH 8.0.Theassayandcontrolcuvettes werebothfilledwith buffer, NaTDC and TLL. cis-PnA was added afterwards intotheassaycuvetteandthedifferentialabsorptionspectra were recorded. Fluorescence spectroscopy Fluorescence measurements were carried out at 29 °C under constant stirring using a SFM 25 spectrofluorimeter from Kontron Instruments and a 3.5-mL quartz cuvette (optical path length 1 cm). During all the fluorescence measurements, the optical density was <0.1 in the spectral range between 280 nm and 500 nm to avoid inner filter effect. Two types of fluorescence experiments were performed. Titration of cis-PnA at various NaTDC concentrations at pH 5.0. The cuvette was filled with buffer containing NaTDC at a given concentration. cis-PnA was then added to the cuvette and a fluorescence emission spectrum was recorded at an excitation wavelength of 320 nm by scanning at an emission wavelength ranging from 350 nm to 500 nm. FRET experiments.TLL(wild-typeormutant)wastitrated at pH 5.0 or pH 8.0 by adding increasing amounts of cis-PnAin thepresenceandabsence of NaTDC.The excitation wavelength was set to 280 nm and the emission wavelength ranged from 300 nm to 500 nm. The accessibility of tryptophan to cis-PnA was estimated by measuring the quenching of the TLL fluorescence effected by cis-PnA, according to the Stern–Volmer equa-tion [25]: F0 ˆ 1 ‡ Ksv‰QŠ …1† where F0 and F are the fluorescence emission intensities in the absence and in the presence of a quencher, respectively, [Q] is the molar quencher concentration and KSV is the Stern–Volmer quenching constant. KSV is appropriate for collisional quenching in which binding is not involved. However, the Stern–Volmer equation fits well our experi-mental results, even though binding is clearly involved. Consequently, KSV will be replaced by ÔKSVÕ. Protein crystallization and crystallography TLL(S146A) solution was washed several times in 10K Centricon in 10 mM Tris/HCl pH 8.0 buffer and concen- Ó FEBS 2002 Lipase binding to lipid, FRET and structural study (Eur. J. Biochem. 269) 1615 trated up to 20 mgÆmL)1. Crystallization trials were performed using the hanging drop technique at 291 K. Screening for the crystallization conditions was performed simultaneously at pH 8.0 (0.1 M Tris/HCl buffer) and pH 5.0 (0.1 M acetate buffer). OA was used instead of cis-PnA for crystallization experiments to avoid oxidation during thecrystallization.OAwasdissolvedin iso-propanol and mixed in this form with a protein sample at a 5 : 1 molar ratio (OA/lipase). After a 1-h incubation, the resulting precipitate was removed by centrifugation in a Sigma Eppendorf centrifuge (5 min, 18 000 g) and the remaining protein was used in the crystallization experi-ments. NaTDC was added to the crystallization trials separately at a concentration of 10 mM. Crystals were flash frozen in the liquid nitrogen and characterized in-house on a Rigaku RU200 rotating anode source (k ˆ 1.5418 A), MAR Research 345 imaging plate scanner, Osmic focusing mirrors and Oxford Cryosystem set at 120 K. The X-ray data were subsequently collected at the ESRF in Grenoble on the MAR Research CCD detector at 100 K, processed with DENZO and scaled and merged with SCALEPACK [26]. The structure was determined using the Molecular Replacement method. The lid was removed by molecular modelling in QUANTA to get a model for molecular replacement (lipase minus lid). The AMORE software program [27] was used and the wild-type TLL structure [14] (minus the lid) was used as a model. The structure was refined using maximum likelihood techniques with REFMAC [28]; other calculations were carried out using the CCP4 suite of programs (Collaborative Computational Project, Number 4, 1994). Electron density map inspection, model building and analysis were carried out with the X-FIT options of the QUANTA software program (Molecular Simulations Inc.). Fig. 1. Fluorescence Emission spectra of TLL (––) and UV absorption spectraofcis-PnA(- - -).TLLandcis-PnAconcentrationswere0.8 lM and 10 lM, respectively. The buffer used was 10 mM acetate (pH 5.0) 100 mM NaCl, 20 mM CaCl2 or 10 mM Tris (pH 8.0) 150 mM NaCl, 21 mM CaCl2, 1 mM EDTA. NaTDC concentration was 1 mM. The excitation wavelength used to obtain the fluorescence emission spectra was 280 nm. RESULTS Absorption spectroscopy The UV absorption spectrum of cis-PnA was determined in an ethanolic solution (95%) and found to be identical to that obtained by Sklar et al. [19]. As cis-PnA is prone to oxidation, the absorption spectrum of the stock solution was checked regularly and no changes in the cis-PnA absorption spectra were observed in the ethanolic solution upon storage. The UV absorption spectrum of cis-PnA at pH 5.0 and pH 8.0 as well as the fluorescence emission spectrum of TLL (excited at 280 nm, at pH 5.0) in the presence of 1 mM NaTDC are shown in Fig. 1. At pH 5.0, the cis-PnA UV absorption spectrum overlapped the TLL emission spec-trum in the 290–380 nm wavelength range, whereas no overlap can be observed at pH 8.0. No significant changes in the TLL emission spectrum were detected between pH 5.0 and pH 8.0 (data not shown). TheeffectsofNaTDContheUVabsorptionspectrumof cis-PnA at pH 5.0 are shown in Fig. 2. In the absence of NaTDC, the cis-PnA solution was slightly turbid. As soon as the NaTDC concentration reached at least 1 mM, the solution became optically clear changing simultaneously the absorption spectrum of cis-PnA. Three main absorption peaks appeared at 298 nm, 304 nm and 326 nm and increased in proportion to the NaTDC concentration. This Fig. 2. Effects of various NaTDC concentrations on the UV absorption spectra of a solution of cis-PnA. The cis-PnA concentration was kept constant at 10 lM. Buffer was 10 mM acetate (pH 5.0) 100 mM NaCl, 20 mM CaCl2. (- - -) 0 mM NaTDC, (– - –) 1 mM NaTDC, (– –) 2 mM NaTDC, (––) 4 mM NaTDC. The schematic diagram on the right illustrates the experimental protocol used. increase in the attenuence of cis-PnA leveled off at NaTDC concentrations above 4 mM. Fluorescence spectroscopy NosignificantNaTDCfluorescencewasrecordedunderour experimental conditions. The excitation and emission spec-tra of cis-PnA were recorded at various NaTDC concen-trations at pH 5.0. The maximum of the excitation and the emission spectra were found to occur at 320 nm and 410 nm, respectively. In order to estimate the critical micellar concentration (CMC) of NaTDC, the relative fluorescence intensity (RFI) of cis-PnA at 410 nm (excita-tion wavelength at 320 nm) was measured as a function of the NaTDC concentration at pH 5.0. At NaTDC concen-trations lower than 1 mM, the fluorescence of cis-PnA was 1616 S. Yapoudjian et al. (Eur. J. Biochem. 269) Fig. 3. FRET between TLL and cis-PnA. The numbers refer to the values of the molar ratio R of cis-PnA to TLL. In all the assays, the excitation wavelength was 280 nm. The dotted line corresponds to the fluorescence emission spectra of cis-PnA (1 lM) recorded in the absence of TLL under the same experimental conditions. The dashed line corresponds to the arithmetic sum ofthe TLL and cis-PnA spectra recorded separately. The correlation between quenched tryptophan RFI (325 nm) and sensitized RFI of cis-PnA (410 nm) is presented in Fig. 4. TLL concentration was 0.8 lM and cis-PnA concentration varied from 0 to 1 lM. For the sake of clarity, only spectrum corres-pondingto three cis-PnA concentrations (0,0.4 and 0.8 lM)are shown (plain lines). The NaTDC concentration was 1 mM. Buffer was 10 mM acetate (pH 5.0) 100 mM NaCl, 20 mM CaCl2. negligible. The RFI increased in parallel with the rise in the NaTDC concentration above 1 mM. This increase leveled off at NaTDC concentrations higher than 4 mM (data not shown). The results of the FRET recordings obtained between TLL and cis-PnA, at wavelengths ranging from 300 to 500 nm in the presence of NaTDC at pH 5.0, are presented in Fig. 3. As the molar ratio (R) between cis-PnA and TLL increased, the RFI decreased at wavelengths ranging from 300 to 380 nm and increased simultaneously at wavelengths ranging from 380 to 500 nm. From the data presented in Fig. 3, the decrease in RFI (%), measured at the maximal emission wavelength (kmax), aswellastheincreaseofRFI(%),measuredat410 nm,asa functionofcis-PnAconcentrationarepresentedinFig. 4.A goodquantitativecorrelationbetweenincreaseanddecrease of RFI as a function of increasing concentration of cis-PnA can be seen. Furthermore, a plateau value is reached when one molecule of TLL is added to one molecule of cis-PnA (R ˆ 1). Similar curves as those presented in Fig. 4 were also obtained for TLL(S146A), TLL(W117F, W221H, W260H) and TLL(W89L) (data not shown). Similar FRET experiments were also performed with TLL, TLL(S146A), TLL(W117F, W221H, W260H), TLL(W89L) and cis-PnA in the presence and absence of NaTDC(Fig. 5).InthepresenceofNaTDC,theFRETwas observed between TLL, TLL(S146A), TLL(W117F, W221H,W260H),TLL(W89L)andcis-PnA.Intheabsence of NaTDC, the FRET was negligible. Surprisingly, in the absence of NaTDC, a clear-cut quenching process was observed only with TLL(S146A) and TLL(W89L). Similar experiments were performed at pH 8.0, in the presence of Ó FEBS 2002 Fig. 4. RFI decrease (d) at kmax as well as RFI increase (s) at k410 nm as a function of cis-PnA concentration. Data from Fig. 3. Fig. 5. FRET between TLL (and its mutants) and cis-PnA in the presenceandabsenceofNaTDC. Theproteinconcentrationwas0.8 lM and the cis-PnA concentration was varied stepwise from 0 to 1 lM (0, 0.2, 0.4, 0.8, 1 lM). Excitation wavelength: 280 nm. Buffer pH 5.0 as in Fig. 2. The data in the graph at the uppermost left hand corner are identical to those shown in Fig. 3. Ó FEBS 2002 Lipase binding to lipid, FRET and structural study (Eur. J. Biochem. 269) 1617 Table 1. Effects of NaTDC (1 mM) and/or cis-PnA (1 lM) on kmax (nm) of the RFI of TLL and its mutants. Data from Fig. 5. Buffer was 10 mM acetate (pH 5.0) 100 mM NaCl, 20 mM CaCl2. The protein concentration was 0.8 lM and the excitation wavelength was 280 nm. Protein (0.8 lM) TLL TLL(S146A) TLL(W117F, W221H, W260H) TLL(W89L) [NaTDC] ˆ 0 mM [PnA] ˆ 0 lM 326 329 335 313 [PnA] ˆ 1 lM 326 328 335 312 [NaTDC] ˆ 1 mM Dkmax [PnA] ˆ 0 lM 0 322 )1 324 0 324 )1 313 [PnA] ˆ 1 lM Dkmax 315 )7 310 )14 304 )20 311 )2 NaTDC (data not shown). Quenching was observed only between TLL(S146A), TLL(W89L) and cis-PnA. The maximum fluorescence emission wavelengths (kmax) ofTLL,TLL(S146A),TLL(W117F,W221H,W260H),and TLL(W89L)(excitation at280 nm, pH 5.0)withor without NaTDC, in the presence or absence of cis-PnA are summarized in Table 1. In the absence of cis-PnA, the addition of NaTDC led to a blue shift of the kmax of all the lipases tested, except for TLL(W89L). Furthermore, in contrasttowhatoccurredwithTLL(W89L),theadditionof cis-PnA in the presence of 1 mM NaTDC also led to a significant blue shift in the case of TLL, TLL(S146A) and TLL(W117F, W221H, W260H). It is worth noting that TLL(W89L)displayedno significantblue shiftunder anyof the experimental conditions tested. Stern–Volmer plots for the fluorescence quenching of TLL (mutants) by cis-PnA were calculated from the data presented in Fig. 5, in the presence of 1 mM NaTDC (data not shown). The ÔKSVÕ constants calculated for TLL, TLL(S146A), TLL(W117F, W221H, W260H) and TLL(W89L) were 3.2.106, 4.6.106, 3.4.106 and 1.5.106 M)1, respectively. X-ray crystallography Good X-ray quality crystals of TLL(S146A) were obtained in the presence of OA in 0.1 M Tris/HCl pH 8.0 buffer, 10 mM NaTDC, 25% w/v poly(ethylene glycol) 5K MME, 25 mM MgCl2. Crystallization at pH 5.0 and control setups were unsuccessful under similar conditions with wild-type TLL. Crystals of the TLL(S146A) mutant were found to belong to the P21212 space group and to have two molecules in the asymmetric unit, a packing density of 2.64 A3ÆDa)1 and a solvent content of 53%. The final X-ray data are 97.8% complete up to 2.20 A resolution (96.2% in the 2.28–2.20 resolution shell) with an overall Rmerge of 0.075 (0.44), I/r(I) of 12.2, and a mean multiplicity of 3.2 observations per reflection. The final model has a crystal-lographic factor of 21.4 and a Rfree of 23.9 against all reflections in the resolution range of 20–2.20 A. The overall root mean square deviations (rmsd) from geometrical ideality are 0.009 A in bond lengths, 1.3° in bond angles, and 1.2 A2 for the DB between bonded atoms. This model is composed of all the atoms of all the residues between E1 and L269 in the case of molecule A (chain A) and molecule B (chain B), with a rmsd of 0.17 A between the corres-ponding Ca atoms of molecule A and B. However, due to the high mobility and the resulting lack of clarity of the electron density maps, occupancies of only a few residues were set at zero during the refinement procedure and consequently in the final model as well. This was the case in particular with loop 24–44 in molecule B and few residues of this loop in molecule A. 262 water molecules were identified and refined. Both molecules have open (Ôfully activeÕ) conformations, as discussed previously [15]. After satisfactory convergence of the refinement of the protein andwatermolecules,theremainingpositiveelectrondensity in the surroundings of the active site cavities was analyzed. This madeitpossibleto model andrefine thefull-lengthOA moleculeintheactivesiteofmoleculeB.Dueto theresidual electron density present in the active site of molecule A, the modeling of the ligand was restricted to its carboxylic group and the alkyl chain between C2 and C9. The remaining atoms of the OA in molecule A, C10–C18, were included in the final protein model for the sake of overall clarity but their occupancies were set to zero, as the electron density of theseatoms was negligible.The OAcompoundwas trapped in the active site of molecule B in an unexpected manner (Fig. 6A); it was rotated by 180° with respect to the main sn-1 TLL alkyl chain binding site [14,15], which is referred to here as antisn1. Structural studies of fatty acids bound to human serum albumin also revealed this unexpected behaviour [29]. The OA carboxylic group of lipid molecule is thrust deeply into this alternative binding cleft of molecule B, where it is anchored via hydrogen bonds between its carboxylic group and the carbonyl oxygen of N92 (2.7 A) and NE2 of H110 (Fig. 6A). The C9–C10 cis double bond lies near the bcarbon of A146 ( 3.0 A), causing this alkyl chain to bend and become wrapped around the W89 residue of the lid, and the C18 carbon is finally wedged between CD1 of I255 and CH2 of W89. The partially defined electron density map of the OA in molecule A binding site cavity was used to model the carboxylic group lying on the top of A146, which is stabilized by a hydrogen bond with the carbonyl oxygen of this residue(2.8 A)andtheNE2 atomofH256 (2.8 A).The fact that C2-C9 carbon atoms of the lipid occupy the sn-1 position in the active site indicates that the lipid binds according to the Ôconventional modeÕ in this molecule. The location oftheremaining OAatomsis notveryclear, dueto the very weak electron density but the position of the first atom of the cis C9–C10 bond was used to model the remaining part of thsis moiety in a similar manner to the model of one of the alkyl chains in the TLL complex with di-dodecyl phosphatidylcholine [15] (not presented in Fig. 6B for the sake of clarity), and with dodecyl phospho-nateinhibitor[14],whichoccupiesthemainsn-1bindingsite as depicted in Fig. 6B. ... - tailieumienphi.vn