Tài liệu Báo cáo khoa học: Solution NMR structure of five representative glycosylated polyene macrolide antibiotics with a sterol-dependent antifungal activity doc

Solution NMR structure of five representative glycosylated polyene macrolide antibiotics with a sterol-dependent antifungal activity Laurent Volpon and Jean-Marc Lancelin Laboratoire de RMN Biomole ´ culaire associe ´ au CNRS, Universite ´ Claude Bernard – Lyon 1 and Ecole Supe ´ rieure de Chimie Physique & Electronique de Lyon, Villeurbanne, France Glycosylated polyene macrolide antibiotics, as nystatins and amphotericins, are amphiphilic structures known to exert antifungal activity by disrupting the fungal cell membrane, leading to leakage of cellular materials, and cell death. This membrane disruption is strongly influenced by the presence and the exact nature of the membrane sterols. The solution structures of five representative glycosylated members, three tetraenes (pimaricin, nystatin A1 and rimocidin) and two heptaenes (candidin and vacidin A) have been calculated using geometric restraints derived from 1 H-NMR data and random searches of their conformational space. Despite a different apparent structural order, the NMR solutions structure indicate that the hydroxyl groups all clustered on one side of the rod-shaped structures, and the glycosyl moieties are structurally conserved both in their conforma- tion and their apparent order. The molecular structures afford an understanding of their selective interaction with the membrane sterols and the design of new polyene macrolides with improved activities. Keywords: antifungal antibiotics; polyene macrolides; sterol- dependant antibiotics; NMR solution structure; 1, 3-polyols. The vast family of polyenes antibiotics [1,2] includes amphiphilic compounds mostly produced by Streptomyces species with potent antifungal properties. Polyene macro- lides are of an authentic clinical value for efficient therapies against animals, and human infectious diseases caused by pathogenic fungi. In particular, nystatin A1, amphotericin B, and pimaricin (natamycin) are the most common polyene macrolides used for the treatment of fungal infections. Due to its particular low toxicity, pimaricin also has been used for a decade as a food preservative [3,4] allowed in the European Union (additive E235) and USA for preserving foods from mold contamination and possible inherent risks of mycotoxin poisoning. The polyene macrolides target the cytoplasmic membranes of fungi where they interact selectively with ergosterol, causing a major disorganization of the membrane structure [5] leading to the leakage of cellular materials and in turn the cellular death. Depending on their molecular structures, polyene macrolides have a more or less toxicity, in part due to a residual interaction with cholesterol in mammalian cyto- plasmic membranes. This gives to polyene macrolides therapies undesired hemolytic and nephrolytic side-effects. Other relevant effects assigned to some polyene macro- lides, such as antiviral properties against several groups of enveloped viruses [6,7] or stimulation of the immune response at lower concentrations [8,9], have been also reported. These activities make of polyene macrolides a source of lead structures for the engineering of future molecules with improved medicinal purposes. In parti- cular, the gene clusters involved in the biosynthesis of pimaricin in S. natalensis [10,11] and nystastin in S. nour- sei [12] have been recently cloned and new models for their biosynthetic pathways been proposed. These new insights make bioengineering possible for new polyene macrolides in addition to chemical synthesis. Despite their discovery over 50 years ago, the under- standing of the selective affinity of polyenes macrolides for sterols in biomembranes has yet no experimental molecular explanation at atomic resolution. The con- formational analysis of different members of the polyene family is one important step essential in understanding their structure-to-activity relationships. Three-dimen- sional structures of only three polyene macrolides of disparate nature and activity, have been described to date. Amphotericin B [13,14] and roxaticin [15] were solved by crystallography, while filipin III was solved using solution NMR [16]. Full stereochemical information (with the exception of one chiral center at position 42 of the vacidin A side chain, Fig. 1) are available for at least five polyene macrolides that belong to a group of polyenes specifically glycosylated by mycosamine, a hexose of the D -series. The glycosylation by an amino sugar occurs near a carboxylic acidic function of the macrolide, so that these polyene macrolides are zwiter- ionic in addition to being amphiphilic. Nystatin A1 was the first polyene macrolide discovered [17]. Its covalent struc- ture (without stereochemistry) was confirmed in 1970 [18] and 1971 [19]. Pimaricin, or natamycin [20], was isolated in 1957 [21] and its covalent structure was established by Golding et al. [22]. Rimocidin from S. rimosus was reported in 1951 [23] and its covalent structure finally described in 1977 [24]. Vacidin A, one of the main components of the aureofacin complex from S. aureofaciens, belongs to the Correspondence to J M. Lancelin, Laboratoire de RMN Biomole ´ culaire, Universite ´ Claude-Bernard – Lyon 1, Domaine Scientifique de La Doua, CPE – Lyon, 43, boulevard du 11 Novembre 1918, F-69622 Villeurbanne cedex, France. Fax/Tel.:+33472431395, E-mail: lancelin@hikari.cpe.fr Abbreviation: ROE, rotating frame Overhauser effect. (Received 11 March 2002, revised 17 July 2002, accepted 23 July 2002) Eur. J. Biochem. 269, 4533–4541 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03147.x aromatic macrolide group [25]. Finally, candidin is a main component of the antibiotic complex produced by S. viridoflavus [26]. These five polyene macrolides have a 26- to 38-membered macrolactone ring, containing a polyol and a polyene part, which is the origin of their amphiphilic nature, and a D -mycosamine sugar (Fig. 1). Asymmetric centers of these five glycosylated polyenes were character- ized by of NMR spectroscopy and stereo-controlled organic synthesis for nystatin A1 [27,28], pimaricin [29,30], rimo- cidin [31], vacidin A [32] and candidin [33]. We took the advantage of the complete knowledge of the stereochemical information of these five polyene macrolides to study their conformation in solution, using NMR and molecular modeling protocols used to explore the confor- mation space of biopolymers [34]. We report herein, the first comparative solution NMR structures of these five repre- sentative 26–38-membered polyene macrolides glycosylated by D -mycosamine. MATERIALS AND METHODS NMR experiments Nystatin A1 sample was obtained from Dr C. Cimarusti, The Squibb Institute for Medical Research (Princeton, New Jersey, USA) [35] and rimocidin (Pfizer Lot #4157-47-2) from Prof Kenneth L. Rinehart, University of Illinois (Urbana-Champaign, Illinois, USA). The antibiotic solu- tions were prepared under dry argon in methanol-d 4 at 3–5 m M concentration. All NMR spectra were recorded at 25 °C on a Bruker Avance DRX 500 spectrometer ( 1 H ¼ 500 MHz) using a 5-mm ( 1 H, 13 C, 15 N) triple- resonance probe head, equipped with a supplementary self shielded z-gradient coil. Spectra were processed using Bruker XWINNMR and GIFA V.4 [36] software. Homo- nuclear two-dimensional spectra DQF-COSY [37], TOCSY (HOHAHA) [38,39] and ROESY [40,41], were recorded with a 1.5-s recovery delay in the phase-sensitive mode using the States-TPPI method [42] as data matrices of 512 (t 1 ) · 1024 (t 2 ) complex data points. Mixing times of 80 ms for TOCSY and 250 ms for ROESY spectra were used. The spectral width in both dimensions was 3500 Hz. The data were apodized with shifted sine-bell and Gaussian window functions in both F 1 and F 2 dimensions after zero- filling in the t 1 dimension to obtain a final matrix of 1024 (F 1 ) · 1024 (F 2 ) real data points. Chemical shifts were referenced to the solvent chemical shift (CHD 2 OD,d ( 1 H) ¼ 3.31 p.p.m.). For heteronuclear spectroscopy, phase-sensitive 13 C- heteronuclear single quantum coherence [43] were recorded with a 1.5-s recovery delay using the echo-antiecho method [44]. The coherence pathway selection was achieved by applying pulsed-field gradients as coherence-filters [45,46]. The FID was collected as a data matrix of 512 (t 1 , 13 C) · 1024 (t 2 , 1 H) complex data points and 150 scans per t 1 increment. Spectral widths were 3500 Hz in F 2 and 17450 Hz in F 1 with carrier frequencies at 3.7 and 70 p.p.m., respectively. For the other three polyenes, the NMR data at 1 H ¼ 300 MHz were taken from the literature where complete 1 H-NMR assignment and experimental restraints are available. Pimaricin was studied in methanol-d 4 [29,30], candidin in a methanol-d 4 /pyridine-d 5 /DMSO-d 6 (2:2:1, v/v) mixture [33], vacidin A in a pyridine-d 5 /methanol-d 4 (9 : 1, v/v) mixture [32]. Experimental NMR restraints For nystatin A1, pimaricin, rimocidin and vacidin A, all the interproton-distance restraints between non J-coupled pro- tons, are derived from the two-dimensional homonuclear ROESY experiments. Interproton restraints were classified into three categories. Upper bounds were fixed at 2.8, 3.3 and 4.0 A ˚ for strong, medium and weak correlations, respectively. For candidin, each of the ROE correlations were considered as weak correlations as no information concerning the ROEs relative intensity were given [32]. A lower bound was fixed at 1.8 A ˚ , which corresponds to the sum of the hydrogen van der Waals’ radii. The intensity of a H i ) H i+2 ROEs within the polyene part was considered as reference intensity for strong correlations [29,30]. Pseudo atom corrections [47] of the upper bounds were applied for Fig. 1. Molecular structures. (A) Pimaricin (B) nystatin A1 (C) rimo- cidin (with R 1 :CH 2 –CH 3 ;R 2 :CH 2 –CH 2 –CH 3 ) (D) candidin and (E) vacidin A. R or S absolute configurations are indicated for asymmetric centers. Carbons are numbered according to their position in the macrolide sequence. Primed indices are assigned to the D -mycosamine glycoside on the right. 4534 L. Volpon and J M. Lancelin (Eur. J. Biochem. 269) Ó FEBS 2002 distance restraints involving the unresolved methylene and methyl protons (+1 A ˚ ). For nonstereospecifically assigned but spectroscopically resolved diastereotopic methylene protons, the interproton distances were treated as single (Ær )6 æ) )1/6 average distances. When possible, H–C–C–H dihedral angle were restrained to dihedral domains accord- ing to the different 3 J HC,CH coupling constants measured using optimized Karplus dihedral relations [16]. When different or very large domains were deduced, some of them could be further restricted from intermediate structure calculations without dihedral restraints. If the resulting models with acceptable energy (see Results) gave a parti- cular dihedral value compatible with the 3 J HC,CH ,the corresponding restraint was applied in a narrower domain. The smallest final dihedral domains were not more restricted than an arbitrary value of ± 20° (Table 3) in case of a correct match between the dihedral angles and the measured couplings. If the dihedral value in the intermediate models was too dispersed, no further restriction was applied. Structure calculations Models were calculated using the X - PLOR software version 3.851 [48] as previously described [16]. Initial atomic coordinates and structure files for each polyene macrolides were generated step by step (given as supporting informa- tion) from the X - PLOR libraries and topology files of different parts of other molecules taken from the Protein Data Bank [49]. For each molecule, the atoms involved in of the lactone function (C–CO–O–C) were maintained in a plane. The hemi-ketal 6-membered rings of the macrolides were maintained in a chair conformation by definition of suitable improper angle restraints. The results were visu- alized using the program MOLMOL version 2.4 [50]. Starting from 30 randomized coordinates, the sampling of the conformational space was performed following a simulated annealing protocol (random SA) proposed by Nilges et al. [51]. The simplified allhdg.pro force field of X-PLOR was used. The nonbonded van der Waals’ interactions were represented by a simple repulsive quadratic term [34,51]. The experimental distance restraints were represented as a soft asymptotic potential, and electrostatic interactions were ignored. The force constant associated with the distance restraints was kept to 50 kcalÆmol )1 ÆA ˚ )2 through- out the protocol. One cycle of random SA consisted of 1500 steps of 3 fs at 1000 K followed by 3000 cooling steps of 1 fs from 1000 K to 100 K. At the end, each structure was subjected to 1500 steps of conjugate gradient energy minimization. RESULTS NMR assignments of nystatin A1 and rimocidin in methanol- d 4 The structure-specific assignment of the 1 Hand 13 C resonances of nystatin A1 and rimocidin in neat meth- anol-d 4 (Table 1) were carried out based on the identifica- tion of various unambiguous resonances. These signals were used as starting points for the complete assignments. In particular, for nystatin A1, starting points were: (a) the well- defined scalar correlations between the two methylene protons at C28 and C29 with their neighboring CH (Fig. 2); (b) the narrow resonance of the anomeric proton H1¢ of the D -mycosamine which is located near d 4.6 p.p.m., and weakly dependent upon the solvent or polyene macrolide nature (Fig. 3); and (c) methyl, ethyl or propyl resonances in the aliphatic region (d 0.95–1.25 p.p.m.) located near the lactone function. NMR-derived geometric restraint In addition to the regular H–C–C–H dihedral restraints derived from the 3 J coupling constants, a particular geometric restraint could be applied for the structure calculation of pimaricin. Indeed, a long-range coupling constant 4 J HO9,H10a ¼ 0.5–1 Hz was already non ambigu- ously assigned [30] to a specific coplanar disposition of the H10a–C10–C9–O9–H9 atoms in the so-called W-arrange- ment [52]. The dihedral angle restraints derived from NMR data in neat methanol for nystatin A1 and rimocidin are summarized in Table 2. Due to some spectrally degenerate proton resonances in the polyene and the polyol regions, complete extraction of the J coupling constants and the ROE information was not possible. The total number of interproton distance restraints derived from the NMR data was 21, 22, 25, 20 and 50 distance restraints for pimaricin (data from [30]), nystatin A1 (this study, Table 1), rimocidin (this study, Table 1), candidin (data from [33]) and vacidin A (data from [32]), respectively. Structure calculations for the five glycosylated macrolide polyenes From the 30 structures calculated, 21, 25, 29, 25 and 29 were retained, respectively, for pimaricin, nystatin A1, rimocidin, candidin and vacidin A on the basis of their low experimental and nonexperimental potential energies (F total <12kcalÆmol )1 ). The models had no ROE viola- tions greater than 0.1 A ˚ nor dihedral violations greater than 5°. Structural statistics are given in Table 3. Structural analysis of pimaricin The superposition for a minimum rmsd of the pimaricin heavy atoms led to a single type of main-chain conforma- tion (Fig. 4A, left). This is likely due in part to the particular macrolactone ring of pimaricin, which is the smallest of the series studied here. This can afford an intrinsic constraint leading to a single conformer under the NMR restraint. The lactone function with the conjugated C2–C3 double bond, the C4–C5 epoxide function, the chair conformation of the C9–C13 heterocycle and the conjugated tetraene C16–C23, altogether form a tightly constrained molecular topology. This topology, in addition to the experimental restraints deduced from ROEs (Table 1) and 3 J information (Table 2) give a very high apparent structural order. The analysis of the model ensemble indicates, a possible hydrogen bond between the hydrogen of OH9 (H O 9) and the oxygen of OH7 (O H 7) which can contribute to the stabilization of the C7–C9 region of the macrolide. Structural analysis of nystatin A1 The spectral degeneracy of 1 H resonances in the polyol as well as in the polyene parts, precluded the derivation of Ó FEBS 2002 NMR structure of glycosylated polyene antibiotics (Eur. J. Biochem. 269) 4535 structural restraints within and between these two structural regions. Therefore, the 25 final models of nystatin A1 appeared well ordered in only two regions: the C11–C27 which is used for the superposition for a minimal atomic rmsd in Fig. 4B (left) and in Table 3, and the C30–C1 segment near the lactone function. An apparent strong structural and local disorder is present in the C2–C10 polyol segment as well as for the two methylenes C28 and C29 at the junction between the tetraene C20–C27 and the diene C30–C33. The absence of experimental restraints in these two regions and the conformational space allowed to the C2–C10 and C28–C29 segments give a strong apparent disorder as shown in Fig. 4. Correlated to this feature, from the six hydroxyl and the carbonyl of the C1–C13 segment of nystatin A1, only the two hydroxyl groups at C11 and C13 are hydrogen bonded in the models (Fig. 4B, left). Other Table 1. 1 Hand 13 C NMR assignments and ROEs of nystatin A1 and rimocidin (in MeOH-d 4 ,25°C). Nystatin A1 Position d 1H a (p.p.m.) d 13C a (p.p.m.) ROEs b Rimocidin Position d 1H a (p.p.m.) d 13C a (p.p.m.) ROEs b 2 2.36 43.1 – 2 2.15 57.8 H2¢¢ d 3 4.17 67.9 – 2¢ (CH 2 ) 1.57; 1.89 23.2 H3 e 4 1.56 44.1 – 2¢¢ (CH 3 ) 0.93 12.1 H3 e 5 3.95 70.4 – 3 4.09 69.4 H24 e 6 1.51 44.6 – 4 2.34; 2.48 45.4 7 3.74 71.5 – 6 2.32; 2.45 49.6 8 1.51 29.9 – 7 2.30; 2.48 9 1.51 29.9 – 8 1.40; 1.65 10 3.29 70.0 – 9 4.11 69.4 H12 d ,H18 c 11 4.03 71.4 H20 c 10 1.28; 1.65 44.9 12ax f 1.58 43.7 H10 c , H14ax c 12ax f 1.28 44.8 H13 e 12eq f 1.86 43.7 H10 d , H14eq c 12eq f 2.01 44.8 H13 c 14ax f 1.28 44.6 – 13 4.26 67.8 H15 c 14eq f 2.02 44.6 – 14 2.15 59.0 15 4.25 67.5 – 15 4.39 67.1 H16ax e , H16eq c H18 d ,H1¢ e ,H2¢ e 16 2.05 60.9 H18ax c 16ax f 1.72 39.0 17 4.26 67.5 H20 d ,H2¢ e 16eq f 2.18 39.0 H1¢ c 18ax f 1.69 39.1 H1¢ d 17 4.49 78.4 H18 d ,H19 c 18eq f 2.10 39.1 – 18 5.92 136.9 19 4.39 78.6 H21 c , H51 c 19 6.14 134.6 20 5.86 135.5 H22 c 20–23 6.32 133.4 21 6.17 132.2 – 24 6.10 134.3 H26 d , H27 d 22 6.28 132.2 – 25 5.62 132.3 H26 d , H27 c 23–25 6.29 132.2 – 26 2.34; 2.49 39.7 26 6.10 132.2 – 27 5.03 74.8 H27¢¢¢ e 27 5.65 135.5 – 27¢ (CH 2 ) 1.54; 1.62 38.5 28 2.13; 2.21 33.2 – 27¢¢ (CH 2 ) 1.40 19.7 29 2.07; 2.17 33.2 – 27¢¢¢ (CH 3 ) 0.94 14.4 30 5.52 132.9 – 1¢ 4.59 98.9 H3¢ c ,H5¢ c 31 5.94 132.2 – 2¢ 4.03 69.0 32 6.00 132.2 – 3¢ 3.19 57.3 33 5.35 135.5 H35 e , H36 e 4¢ 3.42 70.8 H6¢ d 34 2.30 42.1 H36 e , H37 d 5¢ 3.32 74.8 34¢ (CH 3 ) 1.02 17.7 – 6¢ 1.29 17.9 35 3.23 78.6 H34¢ d , H36¢ d , H37 e 36 1.87 41.4 – 36¢ (CH 3 ) 0.95 12.1 – 37 5.16 72.0 – 37¢ (CH 3 ) 1.17 17.2 – 1¢ 4.56 98.9 H3¢ c ,H5¢ c 2¢ 3.98 68.5 – 3¢ 3.14 56.8 – 4¢ 3.30 75.1 H6¢ d 5¢ 3.28 74.0 – 6¢ 1.24 17.3 – a Accuracy of the chemical shifts measured are ± 0.02 p.p.m. and ± 0.2 p.p.m., respectively. b The ROEs connectivities are listed once according to the proton having the lower number. Intensity of the ROEs are strong ( c ), medium ( d ) or weak ( e ). f Refers to the pseudo-axial (ax) or pseudo-equatorial (eq) orientation of these protons relative to the average plane of the macrocycle. 4536 L. Volpon and J M. Lancelin (Eur. J. Biochem. 269) Ó FEBS 2002 hydrogen bonds between the other hydroxyl groups appear only erratically. Structural analysis of rimocidin The small number of restraints that could be applied in the region of C4–C8 segment and for the two aliphatic side chains (Table 2; Fig. 1) yielded a number of different possible conformers in the C27–C8 polyol segment (Fig. 4C, left). The CO groups of C1 and C5 are spread into two major conformations, which are near mirror images each from the other. A hydrogen bond between hydroxyl groups on C9 and C11 (H O 11–O H 9) was detected in 11 out of the 29 models. Structural analysis of candidin As for rimocidin, the polyol part appears the most disordered (Fig. 4D, left). However, for the C1–C7 segment, the two hydroxyl groups on C3 and C5 are, respectively, hydrogen bonded with the CO groups of C1 and C7 in the majority of the models. For the C10–C13 segment, the hydroxyl groups on C11 and C13 form alternatively a H O 13–O H 11oraH O 11–O H 13 hydrogen bond. The absence of information concerning the ROEs relatives intensities (Material and methods; and [33]) is most likely the origin of moderate order of the mycosamine moiety as this region is structurally conserved compared to the other polyene macrolides of this study (Fig. 4, right). Structural analysis of vacidin A The number of experimental restraints available [32] was large for vacidin A, and the 29 final models appear well ordered, except for the aromatic side chain which is spread in random conformations (Fig. 4E, left). As for the four previous polyene macrolides, no experimental information Table 2. Angle restraints deduced (see the text) from J coupling constants for nystatin A1 and rimocidin (500 MHz) in MeOH-d 4 ,25°C. J coupling constants a Angle applied for the calculation nystatin A1 3 J H11-H12ax . ¼ 1 Hz H11–C11–C12–H12ax. ¼ +90° ±20° 3 J H17–H18eq. ¼ 1 Hz H17–C17–C18–H18eq. ¼ )90° ±20° 3 J H18ax–H19 ¼ 1.5 Hz H18ax.–C18–C19–H19 ¼ +90° ±20° 3 J H19–H20 ¼ 8.0 Hz H19–C19–C20–H20 ¼ 180° ±40° 3 J H33–H34 ¼ 8.5 Hz H33–C33–C34–H34 ¼ 180° ±30° 3 J H34–H35 ¼ 8.5 Hz H34–C34–C35–H35 ¼ 180° ±30° rimocidin 3 J H12ax.–H13 ¼ 10.8 Hz H12ax.–C12–C13–H13 ¼ 180° ±20° 3 J H13–H14 ¼ 10.7 Hz H13–C13–C14–H14 ¼ 180° ±20° 3 J H14–H15 ¼ 9.9 Hz H14-C14–C15–H15 ¼ 180° ±20° 3 J H15–H16eq. ¼1 Hz H15–C15–C16–H16eq. ¼ )90° ±20° 3 J H16ax.–H17 ¼1 Hz H16ax.–C16–C17–H17 ¼ +90° ±20° 3 J H17–H18 ¼ 8.5 Hz H17–C17–C18–H18 ¼ 180° ±20° a Accuracy of the J coupling constants measured is ± 0.2 Hz. Fig. 2. Ethylenic-to-aliphatic region (F 2 , F 1 axis) of the phase sensitive DQF-COSY spectrum of nystatin A1, 3 m M in MeOH-d 4 recorded at 500 MHz and 25 °C. The F 1 noise strip at d 3.31 and 4.87 p.p.m. are due to residual signals of the solvent. The two numbers indicated near the cross peaks correspond to the numbering indicated in Fig. 1 of the protons correlated in the F 1 and F 2 axis, respectively. Fig. 3. 1 H NMR spectrum of nystatin A1, 3 m M in MeOH-d 4 recorded at 1 H-500 MHz and 25 °C. Resonance lines are labeled according to the hydrogen numbering indicated in Fig. 1. Asterisks indicate residual signals of the solvent (d 3.31 and 4.87 p.p.m.). Ó FEBS 2002 NMR structure of glycosylated polyene antibiotics (Eur. J. Biochem. 269) 4537 are available about the conformation of hydroxyl groups as they are fully exchanged with the solvent (pyridine-d 5 / methanol-d 4 (9 : 1, v/v) mixture). However, the conforma- tion of the macrolide backbone in the polyol part and the 1,3-syn configuration of the hydroxyl groups from C7 to C15 allow a hydrogen bond network involving H O 7to O H 15orH O 15 to O5 in the majority of the models. The O38-C4 segment is little more disordered and two confor- mations were found for the lactone group, which are mirror images each from the other as for rimocidin. DISCUSSION Solution structures of five polyene macrolides, glycosylated by D -mycosamine have been calculated under NMR- derived geometric restraints. The solubilities of polyene macrolides are too low in water for NMR purpose and the NMR studies were carried out in various polar organic solvents. The five glycosylated polyene antibiotics chosen, are representative of the polyene macrolide family due to their pronounced antifungal activities and their different Table 3. Structural statistics for the pimaricin and nystatin A1. Cartesian coordinate rmsd (A ˚ ) vs. the average geometric structure a pimaricin nystatin A1 rimocidin candidin vacidin A 0.01 (± 0.001) 0.17 (± 0.05) 0.10 (± 0.03) 0.11 (± 0.03) 0.21 (± 0.05) [C1–C25] [C11–C27] [C10–C27] [C12–C37] [C1–C37] Potential energies b in kcalÆmol )1 calculated from X-PLOR – allhdg.pro F total 11.86 (± 0.53) 7.85 (± 0.42) 8.37 (± 0.50) 6.51 (± 0.58) 9.11 (± 0.72) F bond 0.86 (± 0.08) 0.31 (± 0.03) 0.62 (± 0.03) 0.31 (± 0.02) 0.45 (± 0.04) F angle 7.29 (± 0.23) 6.32 (± 0.20) 5.48 (± 0.21) 5.08 (± 0.32) 6.23 (± 0.38) F impr 2.36 (± 0.02) 0.91 (± 0.01) 1.92 (± 0.02) 0.95 (± 0.09) 1.22 (± 0.10) F VDW 0.07 (± 0.02) 0.13 (± 0.19) 0.02 (± 0.06) 0.04 (± 0.03) 0.95 (± 0.07) F roe 0.59 (± 0.14) 0.18 (± 0.12) 0.32 (± 0.21) 0.00 (± 0.01) 0.20 (± 0.12) F cdih 0.66 (± 0.05) 0.00 (± 0.00) 0.01 (± 0.01) 0.12 (± 0.15) 0.05 (± 0.03) a rmsd are calculated for backbone heavy atoms (C) without the side chains (in bracket are given the segment corresponding to the rmsd value). b F bond is the bond-length deviation energy; F angle is the valence angles deviation energy; F impr deviation energy for the improper angles used to maintain the planarity of certain groups of atoms; F VDW is the van der Waals energy function; F roe is the experimental ROE function calculated using a force constant of 25 kcalÆmol )1 ÆA ˚ )2 in the case of the CHARMM22 force field, and F cdih is the experimental function corresponding to the violation of the dihedral angle restraints. In bracket are given the rmsd for certain energetic terms. Fig. 4. Stereoviews of the final NMR models. Left: pimaricin (A), nystatin A1 (B), rimocidin (C), candidin (D) vacidin A (E). Right: Views of the NMR ensembles, seen along the long axis of the polyene with the D -mycosamine front (indicated with an arrow). The bonds of the hydroxyl groups are represented with bold lines. Carbons are labelled according to their numbering indicated in Fig. 1. Models are superposed for a minimum rmsd as indicated in Table 3. 4538 L. Volpon and J M. Lancelin (Eur. J. Biochem. 269) Ó FEBS 2002 macrolactone ring sizes. Two of them, nystatin A1 and pimaricin, are used for human therapies against pathogenic fungi. Pimaricin is the smallest molecule with a 26- membered ring and vacidin A the largest with a 38- membered macrolactone ring. The five NMR ensembles have different apparent struc- tural order: nystatin A1 appears (Fig. 4) the most disor- dered. Due to the lack of direct evidence of conformational averaging, the apparent disorder in the nystatin models cannot be discussed in terms of structural dynamics. The unrestricted two saturated carbons C28 and C29, are however, a possible source of conformational variation by rotation around the axis of the saturated C28–C29 bond. The terminal region C34 to C37 region next to the diene appears well ordered locally and relative to the C30–C33 diene as previously observed [28]. The other four polyene macrolides do not have these saturated carbons within their polyene parts. These polyenes are fully conjugated and are consequently more conformationally restricted. The planar conjugated polyenes are certainly the important elements contributing to the high structural order found in the models of pimaricin, rimocidin, candidin and vacidin A. The extended polyenes constrain the polyol segments of the macrolactone ring to adopt an almost linear staggered conformation to satisfy the ring closure. Altogether, this gives rise to the rod-shaped amphiphilic structure, common to all polyene macrolides. We should note in addition, that the staggered and extended conformation of regular syn 1,3- polyol motifs was proven stable only in apolar media where intramolecular regular hydrogen bonds were found to stabilize this sort of conformation [16,35]. In the presence of competing interactions with polar, protic or aprotic sol- vents, 1,3-van der Waals repulsion dominate and the 1,3- polyols twist to form more stable gauche conformers. The case of the nystatin A1 is interesting regarding its interaction with the sterols and its incorporation into membranes. Unlike to filipin III, a polyene antibiotic belonging to the group of nonglycosylated polyene macro- lides with well-defined conformation [16], the penetration of nystatin [53], as well as amphotericin B [54], into a dilauroylphosphotidylcholine membrane is not possible without the presence of sterols. The conformational variability in the nystatin models gives a better overall solvent accessibility to the hydroxyl groups in the C1–C17 region. We note that this feature correlates to the 10-fold greater solubility in methanol of nystatin A1 relative to amphotericin B. Amphotericin B is structurally very close to nystatin A1. It differs basically by a fully conjugated heptaene segment instead of the potentially flexible polyene motif of nystatin A1. Keeping in mind the greater acces- sibility of the hydroxyls, we hypothesize that nystatin A1 would only insert into a membrane containing sterols after the formation of nystatin-sterols complexes at the mem- brane surface. Such complexes would then yield more rigid molecular edifices, less solvated by water, and in turn more favorable to the antibiotic insertion in the membrane. A common feature appears upon comparison of the five polyenes macrolide solution structures. To illustrate this, we have represented in Fig. 4 (right) the overlays of the different NMR ensembles. The structures are seen from the zwiterionic-head, in the polyene plane common to each antibiotic. From this orientation, we observed that the hydroxyl groups are clustered on one side of the plane, or even on a single axis, parallel to the long axis of the macrolide (pimaricin and vacidin, Fig. 4, right). This correlates also very well with the crystal structure of amphotericin B [14] and the filipin III structure [16]. Another structural character shared by the five macrolides, is the D -mycosamine glycosyl moities always located on the opposite side of the polyene plane. Noteworthy, these common structural features are conserved, regardless of the solvent used to collect the NMR data. The solvents include neat methanol for pimaricin, nystatin A1 and rimocidin ([29,30] and this study); a ternary mixture of methanol/pyridine/DMSO (2 : 2 : 1) for candidin [33]; and a binary mixture of pyridine/methanol (9 : 1) for vacidin A [32]. Clearly, the polyene macrolides share a specific topology of the polar hydroxyls due to a strong intrinsic geometric constraint. This constraint is independent of the solvent and relies on the structure of the macrolide itself. The common struc- tural feature is then likely conserved when the polyenes are transferred from the aqueous compartment to the biomembranes. The five NMR solution structures described here, are important steps in the rationalization of their selective affinity for sterols in biomembranes, and in the design of new polyene macrolides [55] with improved properties. 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Mendes,M.V.,Recio,E.,Fouces,R.,Luiten,R.,Martin,J.F.& Aparicio, J.F. (2001) Engineered biosynthesis of novel polyenes: a pimaricinderivativeproducedbytargetedgenedisruptionin Streptomyces natalensis. Chem. Biol. 8, 635–644. Ó FEBS 2002 NMR structure of glycosylated polyene antibiotics (Eur. J. Biochem. 269) 4541 . Solution NMR structure of five representative glycosylated polyene macrolide antibiotics with a sterol-dependent antifungal activity Laurent Volpon and. activities. Keywords: antifungal antibiotics; polyene macrolides; sterol- dependant antibiotics; NMR solution structure; 1, 3-polyols. The vast family of polyenes antibiotics