Conformation and Dynamics of the Cyclic Lipopeptide Viscosinamide at the Water-Lipid Interface

Abstract

Cyclic lipodepsipeptides or CLiPs from Pseudomonas are secondary metabolites that mediate a wide range of biological functions for their producers, and display antimicrobial and anticancer activities. Direct interaction of CLiPs with the cellular membranes is presumed to be essential in causing these. To understand the processes involved at the molecular level, knowledge of the conformation and dynamics of CLiPs at the water-lipid interface is required to guide the interpretation of biophysical investigations in model membrane systems. We used NMR and molecular dynamics to study the conformation, location and orientation of the Pseudomonas CLiP viscosinamide in a water/dodecylphosphocholine solution. In the process, we demonstrate the strong added value of combining uniform, isotope-enriched viscosinamide and protein NMR methods. In particular, the use of techniques to determine backbone dihedral angles and detect and identify long-lived hydrogen bonds, establishes that the solution conformation previously determined in acetonitrile is maintained in water/dodecylphosphocholine solution. Paramagnetic relaxation enhancements pinpoint viscosinamide near the water-lipid interface, with its orientation dictated by the amphipathic distribution of hydrophobic and hydrophilic residues. Finally, the experimental observations are supported by molecular dynamics simulations. Thus a firm structural basis is now available for interpreting biophysical and bioactivity data relating to this class of compounds.

Identification of the Molecular Determinants Involved in Antimicrobial Activity of Pseudodesmin A, a Cyclic Lipopeptide From the Viscosin Group

“…Since our screening aimed to investigate the molecular determinants of activity at the level of the peptide, we used the response of these most sensitive strains to identify loci of importance for bioactivity, whereas the response recorded for the less sensitive strains was used to assess its generality importance. All biological activity results were confronted with the fold of pseudodesmin A (Figure 4B), complemented with information obtained using NMR on the location and orientation of viscosinamide in DPC micelles (Geudens et al, 2017(Geudens et al, , 2019. While both CLiPs differ by a D-Leu5 to L-Leu5 switch in stereochemistry, they are equally active.…”

“…In DPC/H 2 O solutions of viscosinamide, the amphipathic distribution of residues over opposing faces of the viscosin CLiP fold drives individual molecules to be located near the phosphocholine head groups of DPC molecules. The α-helix orients parallel to the lipid/water interface with the center of mass close to the transition of the polar head-group to the non-polar lipid chain and the hydrophobic moment pointing toward the micelle's core (Geudens et al, 2019). Applying this orientation to pseudodesmin A (Figure 4A) most hydrophobic side-chains oriented toward the lipid interior (Figure 4B), while the polar residues are exposed to the choline head group and are readily solvent accessible (Figure 4B).…”

“…Later work demonstrated that in order to establish this solution structure in DPC micelles, isotope labeled compound was an absolute requirement. Preference was given to production of viscosinamide (D 0 ), because of the high biosynthetic performance of its bacterial producer (DR54), a trait required when producing isotope labeled compounds from minimal media (Geudens et al, 2019). This was not the case for the original Pseudodesmin A producer which is currently unavailable due to third party patent protection issues.…”

“…Such an approach requires an efficient (bio-)synthetic route for production of relevant analogs, unavailable from Nature, whose biological evaluation of activity can then be interpreted in terms of a three-dimensional structure in a biologically relevant context. Here, we present such a study for the viscosin group of Pseudomonas CliPs, building on our previously reported total synthesis route for pseudodesmin A (1, PsdA) and our recently obtained understanding of the structure and dynamics of viscosinamide A at the lipid-water interface using its investigation by NMR in a dodecylphosphatidylcholine (DPC):water solution (Geudens et al, 2019).…”

“…To proceed toward full structure-activity studies, total synthesis must be complemented by knowledge of the CLiP fold within the lipid-water interface. To this end, we determined the solution structure and the dynamics of viscosinamide (L 0 ) in a DPC-water mixture using NMR spectroscopy and Molecular Dynamics simulations (Geudens et al, 2019). We showed that the solution conformation previously available only from organic solutions or in a crystal environment, is fully maintained in DPC micelles.…”