Higher-Level Structural Classification of Pseudomonas Cyclic Lipopeptides through Their Bioactive Conformation

Cyclic lipodepsipeptides (CLiPs) from Pseudomonas comprise a large pool of non-ribosomally synthesized, membrane targeting specialized metabolites with diverse ecological roles and antimicrobial activities. Over the past decades significant efforts have been made to reveal their chemical constitution and configuration thus providing the starting point to establishing structure–function correlations, derive molecular level understanding of their mode of action and ultimately harness their potential in plant biocontrol and clinical applications. The sheer diversity in chemical structures combined with few, scattered reports of three-dimensional structures has been a limiting factor to advance in these areas. Here, we present the solution conformations of eight antimicrobial, non-phytotoxic Pseudomonas CLiPs, each representing a distinct CLiP family including MDN-0066, viscosin, orfamide A, arthrofactin A, tanniamide A, xantholysin A, putisolvin I and entolysin A, obtained using a consistent protocol that combines NMR spectroscopy in membrane mimicking dodecylphosphocholine solutions with all-atom molecular dynamics simulations. All CLiP conformations share a left-handed alpha-helix forming either a stapled (m>5) or catch-pole helix (m≤5) motif, depending on the number of residues in the macrocycle (m). This structural dichotomy was validated through a synthetic analogue of the naturally occurring orfamide A featuring an alternative, more constricted macrocycle. The two motifs define distinct superfamilies encompassing most known Pseudomonas CLiPs thereby offering a new framework for their higher-order structural classification. Correlations between the obtained Pseudomonas CLiP conformations and the organization of their biosynthetic gene clusters were found which support future homology modelling efforts for these metabolites.