Metal‐ion Binding to Host Defense Peptide Piscidin 3 Observed in Phospholipid Bilayers by Magic Angle Spinning Solid‐state NMR

Abstract

Cationic antimicrobial peptides (AMPs) are essential components of the innate immune system. They have attracted interest as novel compounds with the potential to treat infections associated with multi‐drug resistant bacteria. In this study, we investigate piscidin 3 (P3), an AMP that was first discovered in the mast cells of hybrid striped bass. Prior studies showed that P3 is less active than its homolog piscidin 1 (P1) against planktonic bacteria. However, P3 has the advantage of being less toxic to mammalian cells and more active on biofilms and persister cells. Both P1 and P3 cross bacterial membranes and co‐localize with intracellular DNA but P3 is more condensing to DNA while P1 is more membrane active. Recently, we showed that both peptides coordinate Cu2+ through an amino‐terminal copper and nickel (ATCUN) motif. We also demonstrated that the bactericidal effects of P3 are linked to its ability to form radicals that nick DNA in the presence of Cu2+. Since metal binding and membrane crossing by P3 is biologically important, we apply in this study solid‐state NMR spectroscopy to uniformly 13C‐15N‐labeled peptide samples to structurally characterize the ATCUN motif of P3 bound to bilayers and coordinated to Ni2+ and Cu2+. These experiments are supplemented with density functional theory calculations. Taken together, these studies refine the arrangement of not only the backbone but also side chain atoms of an AMP simultaneously bound to metal ions and phospholipid bilayers.

Copper-binding anticancer peptides from the piscidin family: an expanded mechanism that encompasses physical and chemical bilayer disruption

“…Based on the results mentioned above, the bilayer structural perturbations that we observe in the presence of holo-P1 and -P3 are exclusively due to the complexation of the metal ions with the peptides and the complexation affects the interactions of P1 and P3 with the lipid bilayer. In particular, the resulting net loss of one positive charge at the amino end of the peptide upon metallation very likely facilitates the bilayer insertion of the metallated peptides 63 , 64 . Given that metallation more dramatically impacts the IC 50 and membrane conformation of P3 compared to P1, we further examined P3-Cu 2+ within the bilayer and used available 3D structures to characterize its orientation.…”

“…The metal is coordinated by four nitrogens, including the nitrogen from the N-terminus, the two subsequent backbone amide nitrogens, and the δ-nitrogen of His-3 arranged in a distorted square planar coordination geometry 68 , 70 , 71 . The ATCUN motif requires the deprotonation of the three backbone nitrogen sites, and thus metallation is more favorable at basic pH 59 , 63 , 64 . Once bound to Cu 2+ , ATCUN-containing peptides become distinctively pink, a useful property to ascertain their metallation state under changing sample conditions 63 , 64 .…”

“…The ATCUN motif requires the deprotonation of the three backbone nitrogen sites, and thus metallation is more favorable at basic pH 59 , 63 , 64 . Once bound to Cu 2+ , ATCUN-containing peptides become distinctively pink, a useful property to ascertain their metallation state under changing sample conditions 63 , 64 . Given the ability of Cu 2+ to form ROS, synthetic peptides containing the ATCUN motif have been designed to generate radicals and damage harmful cells and pathogens, including in vivo 67 , 69 , 71 – 77 .…”

Metallated Anticancer Peptides: An Expanded Mechanism that Encompasses Physical and Chemical Bilayer Disruption

“…Uncertainty bands in the deuterium profiles (colored bands) were determined using a 68% confidence interval in the Monte-Carlo sampling of the measured structure factors 86 . The inset show representative average conformation of the P3 -helix in the bilayer, using a 3D structure derived from the NMR structure (PDB ID # 6PEZ) 58 and the predicted structure of the Cu 2+ -bound ATCUN motif based on density functional theory calculations 64 . The orientation of the α-helix in the bilayer is derived from the diffraction SLD profiles.…”

Metal–Peptide Complexes as Promising Antibiotics to Fight Emerging Drug Resistance: New Perspectives in Tuberculosis

“…The ATCUN motif is Phe-Phe-His-for P1 and Phe-Ile-His-for P3 and binds Cu 2+ and Ni 2+ ions with high affinity: the α-amine group, the two amide nitrogens of the backbone, and the δ-nitrogen of the third His form the ligand field with a planar coordination geometry [77]. Both P1 and P3 cross bacterial membranes and co-localize with intracellular DNA, but P3 is more condensing to DNA, while P1 is more membrane-active, and the bactericidal effects of P3 are enhanced in the presence of Cu 2+ caused by the formation of radicals (superoxide O 2 , hydroxyl radical HO) that nick DNA [78]. Interestingly, progressive loss of the plasmid supercoiled form pUC19 and an increase of the nicked and linearized forms of DNA were outlined during the time-dependent cleavage of the plasmid incubated with P3 in the presence of H 2 O 2 and ascorbic acid [76].…”