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

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.     

The effects of chromium(III) coordination on the dissociation of acidic peptides

The complexes formed between chromium(III) and synthetic acidic peptides were studied by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion-cyclotron resonance (FT-ICR) mass spectrometer equipped with electrospray ionization (ESI). Neutral peptides and peptides containing one, two, and multiple acidic residues were studied. Formation of [M + Cr-2H]+ occurred for all peptides. Three noteworthy features were found in the CID spectra of [M + Cr-2H]+. The first is that fewer fragment ions were produced from [M + Cr-2H]+ than from [M + H]+. The reason may be that multiple coordination between chromium(III) and carboxylate or carbonyl groups hinders the production of fragment ions by continuing to bind pieces of the peptide to chromium(III) after cleavage of bonds within the peptide. The second feature is loss of CO from [M + Cr-2H]+ and [y(n) + Cr-H]+. A mechanism involving coordination of chromium(III) with carboxylate groups is proposed to rationalize elimination of CO. The third feature is that chromium(III) is retained in all fragment ions, indicating strong binding of the metal ion to the peptides. The complex [M + 2Cr-5H]+ is formed as the peptide chain length and number of acidic residues increases. Longer peptides have more sites to coordinate with chromium(III) and more conformational flexibility. In addition, formation of [M + Cr-2H]+ from AGGAAAA-OCH(3), which has no carboxylic acid groups, suggests that chromium(III) can coordinate with sites on the peptide backbone, albeit in low abundance. In the negative mode, [M + Cr-4H](-) was only found for peptides containing four or more carboxylic acid groups. This is consistent with deprotonated carboxylic acid groups being involved in chromium(III) coordination and with chromium existing in the 3 + state in the gas-phase ions.