Kovács, Benjámin

Martins, José

Ghent University Academic Bibliography

IN THE WAKE OF THE MAIN PRINCIPLE BEHIND THE DESIGN OF NATURAL CYCLIC LIPODEPSIPEPTIDES1 NMR AND STRUCTURE ANALYSIS RESEARCH UNIT, DEPARTMENT OF ORGANIC AND MACROMOLECULAR CHEMISTRY, GHENT UNIVERSITYB. Kovács1, N. Geudens1, José C. Martins13-HDA   L-Leu1 D-Glu2 D-aThr3 D-Val4  L-Leu5  D-Ser6     L-Leu7   D-Ser8   L-Ile9lllll3-HTDA L-Leu1 D-Glu2 D-aThr3 D-Val4  L-Leu5  D-Ser6     L-Leu7   L-Leu8   D-Ser9    D-Val10l3-HDA  L-Leu1   D-Glu2   D-Gln3 D-Val4   L-Leu5 L-Gln6  D-Ser7   D-Val8  D-Leu9  D-Gln10  L-Leu11 L-Leu12 D-Gln13 L-Ile14lll3-HDA   D-Leu1 D-Asp2 D-aThr3 D-Leu4 D-Leu5 D-Ser6     L-Leu7    D-Gln8   L-Leu9   D-Ile10   L-Asp11OOO2: V,O,A6: X viscosin A (V):orfamide B (O):xantholysin A (X):amphisin (A):N-terminusC-terminuscyclization via depsi bondfatty acid9: A8: O,X7: V5: A4: O,X3: V6: V,O,A10: XNr of endocyclic AANr of exocyclic AANr of αL-helical AA Nr of loop AA6Nr of αL-helical AAxantholysin A NMR structure (DCP-d38+water)/CNS+AMBERorfamide BNMR structure (AcN)/CNSviscosin ANMR structure (AcN)/CNS3O, X: +4 exocyclic AA• +1 helical turn!• ring conformationstays constantNr of loop AA104amphisin [8]X-ray structure5V, O, A: ring size changes • loop structure adapts• helix size stays constantOOrigin and primary structure of CLPsCyclic lipodepsipeptides (CLPs) are secondary metabolitestypically produced by Pseudomonas and Bacillus bacterial speciesvia non ribosomal pathways [1]. These biomolecules areconstituted of a fatty acid moiety linked to the N-terminus of apeptide chain which is cyclized by an ester (or depsi) bondformation between its C-terminus and a hydroxyl group cappedside chain of a Ser or Thr residue. Due to the nature of thebiosynthesis, the familiar prevalence of L-amino acids is flawed bythe significant presence of residues maintaining D-configuration.The CLPs are classified depending on the length of the peptidechain and on the size of the cycle. Herein, the eponym membersof viscosins, orfamides, xantholyisins and amphisins will bescrutinized (Fig. 1). It is apparent that the alternation of aminoacids with polar and apolar side chain is a common feature. Istheir order random or purposeful? Figure 1: The primary structures of the presented CLPs. The fatty acid parts (3-HDA = (R)-3-hydroxydecanoic acid and 3-HTDA = (R)-3-hydroxytetradecanoic acid are highlighted by grey, while the amino acid residues are colored according to the polarity of their side chain, i.e. hydrophilic = red, hydrophobic = green. Why CLPs? What are we looking for?CLPs possess a wide range of biological activites: their antifungal, antiviral, anticancer and antibacterial effects [1–3] have already been exploited e.g. in the field ofcrop protection [4]. More importantly, CLPs provide either of the two classes of antibiotics that have been approved for clinical use in the past 30 years [5].The level of bioactivity depends on the mode of action that is inherently determined by the structures of the chosen agent and its target. In case of CLPs themolecular background of the function is not well understood. To start with, the conformation of cyclic lipodepsipeptides is yet poorly explored (the tertiary structureis available for less than 10% of the already identified ~100 CLPs). Thus, an intention of this study is to present and correlate the previously unknown solutionconformation of viscosin A, orfamide B and xantholysin A. Moreover, the effect of gradual ring size extension can be studied systematically by involving amphisin (Fig.1) in the analysis. Such structural comparison spanned over distinct CLP subgroups might lead us to realize the principle behind the design of natural cycliclipodepsipeptides which could be a milestone on the path towards the development of novel efficient pharmaceuticals.How?The peptides were biosynthetized by Pseudomonas strains andsubmitted for HPLC purification afterwards (both conducted locally,at UGent). To gain insight to the conformation at atomic resolution,solution state NMR spectroscopy was applied for collecting 1H–1Hdistances. This data was used as a set of distance restraints whichaided the silico structure determination performed by moleculardynamics simulations using the force fields of the CNS 1.21 [6] andAMBER14 software packages [7] (Fig. 2).Results – Tertiary structureThe three-dimensional structures of the four CLP family representatives are built from the same elements: an αL-helix kicks off from Leu1 and penetrates up to the 4th endocyclic residue (Fig. 2). From then on, it proceeds into loopwhich connects the C-terminal with an OH-capped side chain via a depsi bond. This means that the length of the αL-helixstays the same for viscosin A, orfamide B and amphisin (involving 6 residues for each) and the extension of the ringinfluences the loop size and conformation. The latter is varying in order to maintain the perfect separation of thehydrophobic and hydrophilic side chains what was already established by the helical motif and present for all cycliclipopeptides in question. On the other hand, orfamide B and xantholysin A bear the same ring arrangement in terms ofthe side chain polarity in the respective positions. As a result, their ring and loop conformation are identical. The fourextra exocyclic residues for xantholysin A simply prolong the amphipathic αL-helix with an additional turn opposed toorfamide B. Along with the three-dimensional structures of cyclic lipopeptides containing non-natural amino acids[tolaasins: 2, syringomicins: 9], the herein presented observations confirm the preservation of the amphipathic characterto be a fundamental driving force for the construction of natural CLPs.111Future prospects – Mode of actionConcomitantly, the insertion of the amphipathic cyclic lipopeptides in apolar solvent triggers their self-assembly. Thephenomenon was examined in detail for pseudodesmin A (member of viscosins) which pointed out the occurence oftubular-shaped associations (Fig. 3) [10]. A familiar explanation for the mechanism of action of antimicrobial peptides isthe creation of such pore-like associations which disrupt the plasma membrane and cause the lysis of the cell [11].Obviously, investigating the bioagent alone does not allow to make general assumptions, since the actual processdepends on the chosen target as well.The application of the 15N and 13C labelled version of the peptides would enable to demonstrate the presence anddistinguish between inter- and intramolecular H-bonds [12], thus to reveal the structure of self-assemblies or evencoaggregates of peptides and membrane mimicking detergent micelles/bicelles at high resolution. Such experimentscould take us one step closer to the revelation of the structure–function relationship of CLPs. As the recent efforts hadshown, growing viscosinamide-producer Pseudomonas sp. DR54 in a minimal salt medium prepared by the addition of15NH4Cl successfully yielded the15N-enriched biomolecules in interest, so this method is planned to be accomplished theisotopical labelling of other CLPs as well.ConclusionBy systematically studying the effect of the total peptide chain and/or ring residue number on the overallconformation, we can conclude that the amphipathic character is a crucial factor in the design of natural CLPs,irrespectively of the exact amino acid composition. The 15N and 13C labelled lipopeptides will make the directidentification of intra- and intermolecular H-bonds in self-assemblies/peptide–detergent coaggregates feasible,thus bring more details to light about the mode of action behind the versatile bioactivity of these biomolecules.References[1] Raaijmakers, J.M.; De Bruijn, I. et. al., Mol. Plant. Microb. Interact. 2006, 19 (7), 699–710[2] Jourdan, F.; Lazzaroni, S. et. al., Proteins: Struct., Funct., Genet., 2003, 52, 534–543[3] Pascual, J.; Garcia-López, M. et. al., Syst. Appl. Microbiol., 2014, 37, 412–416[4] Khong, N.G.; Randoux, B. et. al., Commun. Agric. Appl. Bio. Sci., 2013, 78 (3), 479–487 [5] Fishbach, M.A.; Walsh, C.T.; Chem. Rev., 2006, 106, 3466–3496[6] Brunger, A. T.; Nat. Protoc., 2007, 2, 2728–2733[7] Salomon-Ferrer, R.; Gotz, A.W. et. al., J. Chem. Theory Comput., 2013, 9, 3878–3888[8] Sorensen, D.; Nielsen, T.H. et al., Acta Cryst. C, 2001, 57, 1123–1124.[9] Anselmi, M.; T. Eliseo et al., Biochim. Biophys. Acta–Biomembranes, 2011, 1808, 2102–2010. [10] Sinnaeve, D.; Hendrickx, P.M.S. et al., Chem. Eur. J., 2009, 15, 12653–12662[11] Lee, T.; Hall, K.N. et al., Curr. Top. Med. Chem., 2016, 16, 25–39 [12] Cordier, F.; Nisius, L.,; Nat. Protoc., 2008, 3, 235–241Figure 2: The tertiary structures ofthe scrutinized CLPs. In the bottomright corners the amphipathiccharacter is emphasized by coloringthe whole side chains according totheir polarity. For better overview,the fatty acid part is omitted. Theexperimental conditions and thesoftware(s)/force field(s) used forthe structure calculations are alsoindicated.Contact: Benjamin.Kovacs@UGent.be /+32 (0)9 264 44 77Universiteit Gent@ugentGhent UniversityFigure 3: Top: the proposed model forthe self-assembly based on diffusionmeasurements (adapted from [10]).Bottom: the peaks in the 1H spectrumof pseudodesmin A dissolved in CDCl3(15 mM) care uniformly broadened dueto the hindered rotational diffusion.Here, only the aliphatic region isdisplayed. The translational diffusionconstant measured for the assembly(Dt, 1H) and the hypothetical value for amonomer (Dt, m) are also displayed.2.5D-aThr3L-Leu1L-Ile9D-Ser62.0 1.5 1.0 [ppm]Rel. Int.123Dt, 1H = 113.6± 0.3 (μm)2/sDt, m = 561.4 ± 3.0 (μm)2/s1H spectrum at 298.2 K

In the wake of the main principle behind the design of natural cyclic lipodepsipeptides

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In the wake of the main principle behind the design of natural cyclic lipodepsipeptides

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