Kovács, Benjámin

Martins, José

English

Ghent University Academic Bibliography

DIRECT DETECTION OF N-H...O=C H-BONDS IN CYCLIC LIPODEPSIPEPTIDES BY NMR SPECTROSCOPY1 NMR AND STRUCTURE ANALYSIS RESEARCH UNIT, DEPARTMENT OF ORGANIC AND MACROMOLECULAR CHEMISTRY, GHENT UNIVERSITYB. Kovács1, José C. Martins1References[1] Raaijmakers, J.M. 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] Khong, N.G.; Randoux, B. et. al., Commun. Agric. Appl. Bio. Sci., 2013, 78 (3), 479–487 [4] Fishbach, M.A.; Walsh, C.T.; Chem. Rev., 2006, 106, 3466–3496 [5] Pascual, J.; Garcia-López, M. et. al., Syst. Appl. Microbiol., 2014, 37, 412–416 [6] Cordier, F. et. al., Nat. Protoc., 2008, 3, 235–241 [7] Brunger, A. T.; Nat. Protoc., 2007, 2, 2728–2733 [8] Salomon-Ferrer, R.; Gotz, A.W. et. al., J. Chem. Theory Comput., 2013, 9, 3878–3888 [9] Ester, M. et. al., A density-based algorithm for discovering clusters in large spatial databases with noise, AAAI, Portland, OR, 1996, 226–231 Contact: Benjamin.Kovacs@UGent.be /+32 (0)9 264 44 77Universiteit Gent@ugentGhent UniversityPeptideCyclePeptideExocycleFatty acid(R)-3-HDA-L-Leu1-D-Gln2-D-allo-Thr3-D-Val4-L-Leu5-D-Ser6-L-Leu7-D-Ser8-L-Ile9OOOviscosinamide DepsiNH2The state-of-the-art on CLP structureCyclic lipodepsipeptides (CLPs) are secondary metabolites typically producedby Pseudomonas and Bacillus bacterial species via non ribosomal pathways [1].These biomolecules are constituted of a fatty acid moiety linked to the N-terminus of a peptide chain which is cyclized by an ester (or depsi) bondformation between its C-terminus and a hydroxyl group capped side chain of aSer or Thr residue. Due to the nature of the biosynthesis, the familiarprevalence of L-amino acids is flawed by the significant presence of residuesmaintaining D-configuration. Additional peculiarity is the alternation of aminoacids with polar and apolar side chain.The tertiary structure of CLPs is yet poorly understood, however, somecommon features are already obtrusive from this respect. Their peptidebackbone is composed of an α-helix proceeding into a loop which enables thedepsi bond connection. Moreover, the perfect separation of polar and apolarside chains established by the helix is maintained in the loop, which’sconformation seems adapted to preserve the amphipathic character [e.g. 2].3DL-L1D-aT3D-S6L-L7L-I8αL-helixloopPolar side chain: red Apolar side chain: greenmeritamphipathic character !CLP bioactivityCLPs serve as a master biochemical tool box for their bacterial producers; theynot only facilitate bacterial swarming (motility) and biofilm formation [1] butmay take role in the stimulation of the plant immune system what has alreadybeen exploited in the field of crop protection [3]. Moreover, in vitro testing hasrevealed activity against a broad range of microbials, including bacteria, viruses,fungi [1] (non-exhaustive). Actually, cyclic lipopeptides constitute either of the two classes of antibioticsthat have been approved for clinical use in the past 30 years: daptomycin (marketed as CUBICIN®) is a CLPapplied for skin infections caused by Gram-positive bacteria [4]. Furthermore, CLPs as e.g. xantholysin,MD0066 and viscosin were reported to exhibit anticancer effects at a concentration below cytotoxic level[5].Structure – function relationship not well understood!Though it would be crucial en route to the development of novel pharmaceuticals...Aim: to gain more detailed structural information   Direct H-bond detection !Methods: bacterial growth in minimal salt mediumFor direct [N–H...O=C]-type H-bond detection solution state NMR spectroscopy wasused. The experiment in question (modified HNCO [6]) involves 1H, 13C and 15Nnuclei. Unfortunately, the natural abundance of 13C and 15N is very low (1.1% and0.4%, respectively). For the sake of sensitivity, the investigated peptide had to beenriched in 13C and 15N isotopes (a.k.a. isotope labelling). This required to modifyour routinely applied biosynthetic protocol, i.e. the bacteria had to be grown inminimal salt medium instead of the a priori routinely applied King’s B (KB). Tested bacterial strain: viscosinamide producer Pseudomonas sp. DR54KB medium: salts + proteose peptone (N-, C-source ) + glycerol (C-source)M9 medium: salts + NH4Cl (N-source) + glucose (C-source)t/ hrelative viscosinamide productionpeptide harvestafter 24 hM9 not only worked, butprovided more CLP andrestricted the evolution ofother metabolites comparedto KB! Interpretation: bacterialswarming ... [1]two-fold merit• In our group M9 basically replaced KB forstandard growths  more efficient growth,isolation, purification... higher pure CLP yield!• successfully applied for several otherPseudomonas sp. strains since then:e.g. COR55 putisolvin; COR5 entolysin ...• 14NH4Cl15NH4Cl• 12C-glucose U-13C-glucoseincorporationof 13C and 15N13C/15N-labelled viscosinamide !Remark: since 15NH4Cl is $and U-13C-glucose is $$$,their concentrations werefurther optimized.simulated annealingsimulated annealing +MD/cluster analysisExperimental:In silico:1H dimension13C dimensionResults: the H-bond network of viscosinamideThe isolated and purified 13C/15N-labelled viscosinamide was dissolved in AcN-d3 for NMR investigations. TheHNCO experiment indicated the presence of three [N–H...O=C]-type H-bonds.For peptide ’structure elucidation’ in solution state, it is common to collect 1H–1H distance restraints by NOESYNMR experiment which guides a series of simulated annealings [7] to find realistic conformations. Thanks tothe restraints imposed on (N)H and αH positions this method leads to valid backbone conformation, but theC=O bond orientations can be doubtful (see below). We complemented this method with time-dependent MDsimulation [8] as refinement step, and a static conformation was assessed by a self-tailored cluster analysis [9]protocol. The improved in silico result was in perfect agreement with the experimental observations.I: Val4(N–H)HDA (O=C)II: Leu5(N–H)Leu1(O=C)III: Ser8(N–H)aThr3(O=C)132I(cross)/I(ref.)   h3JNC(exp.)h3JNC(calc.)Conclusion and future prospectsWe managed to give accurate picture about the H-bond pattern of a cyclic lipodepsipeptide usingadvanced NMR and MD methods. This work is pioneering in that context, since earlier neither the productionand 13C/15N-labelling using minimal salt medium, nor direct detection of H-bonds had been performed forCLPs. In the future both intra- and intermolecular H-bonds will be identified in a similar fashion forCLP/micelle coaggregates and self-assemblies. To further exploit the advantages of 13C/15N-labelling NMR-wise, isotope-filtered 1H-1H NOESY is planned to be used for the structure calculation of CLP self-assemblies.That would allow the first ever investigation of such supramolecular construction at atomic details.[6][N–H...O=C]I: Val4(N–H)HDA (O=C)II: Leu5(N–H)Leu1(O=C)III: Ser8(N–H)aThr3(O=C)and nothing else!III[N–H...O=C]I: Val4(N–H)HDA (O=C)II: Leu5(N–H)Leu1(O=C)III: Ser8(N–H)aThr3(O=C)IV: Ser6(N–H)Gln2(O=C)V: Leu7(N–H)aThr3(O=C)IIIIIIIIIL1S8aT3L5HDAQ2S6 sidechain OHQ2S6 sidechain OHIVVIVblank positions (     ) in HNCO spectrumaT3L7Q2S6

Direct detection of N-H...O=C H-bonds in cyclic lipopeptides by NMR spectroscopy

https://biblio.ugent.be/publication/8587644/file/8587645

Direct detection of N-H...O=C H-bonds in cyclic lipopeptides by NMR spectroscopy

Abstract

Similar works

Full text

Available Versions

Ghent University Academic Bibliography