Structural Basis of a Temporin 1b Analogue Antimicrobial Activity against Gram Negative Bacteria Determined by CD and NMR Techniques in Cellular Environment

We here report an original approach to elucidate mechanisms of action of antimicrobial peptides and derive crucial structural requirements for the design of novel therapeutic agents. The high resolution structure of TB_KKG6A, an antimicrobial peptide designed to amplify the spectrum of action of Temporin B, bound to <i>E. coli</i> is here determined by means of CD and NMR methodologies. We have also defined, through STD analysis, the residues in closer proximity to the bacterial membrane

NMR characterization of the monomers.

<p>Superimposition of the 2D-TOCSY, expansion of the backbone region, for the g^S and g^OH monomers.</p

CD of single strands.

<p>CD spectra of PNA S (–) and PNA OH (–) in 10 mM Phosphate buffer 100 mM NaCl, 5 mM MgCl₂, pH 7 at 10 µM strand concentration.</p

PNA monomers.

<p>Schematic representation of standard and modified PNA monomers.</p

Flow cytometric analysis of ErbB2 expression on SKBR3 cells.

<p>Cells are treated with different amounts of PNA S and PNA in the presence of lipofectamine. (<b>A</b>) Single result representative of three similar experiments. Not transfected cells (black curve), not transfected and treated cells with an antiErbB2 antibody (green curve), transfected with 10 µM PNA S and treated with an antiErbB2 antibody (red curve) and transfected with 10 µM PNA and treated with an antiErbB2 antibody (blue curve). (<b>B</b>) Histograms were obtained from at least three independent experiments. Data are expressed as percentage of decrease versus control (cells not transfected and treated with an antiErbB2 antibody) ± SE. Statistical significance was carried out by means of the two tailed paired Student’s t test, **p<0.01, *p<0.05.</p

qPCR of ErbB2 and GAPDH genes.

<p>RNA was obtained after transfection of SKBR3 cells with PNA S or PNA at 5 and 10 µM concentration. The experiments were performed in triplicate and repeated at least 3 times. Data indicate the relative gene expression of cells treated with PNA S or PNA versus not treated cells, error bars represent the standard deviation of the mean. Statistical significance was carried out by means of the two tailed paired Student’s t test, *p = 0.02; **p = 0.0006.</p

CD of triplexes.

<p>CD spectra of (PNA S)₂DNAc (–), (PNA)₂DNAc (–) in 10 mM Na Phosphate buffer, 100 mM NaCl, 5 mM MgCl₂, pH 7 at 3 µM triplex concentration.</p

Monomers rotamers.

<p>Representation of the two rotamers found for the sulphate monomer g^S.</p

Oligomers sequences. PNA bases are indicated with lower case letters, DNA bases with upper case letters.

<p>Oligomers sequences. PNA bases are indicated with lower case letters, DNA bases with upper case letters.</p

Sulphate monomers conformation.

<p>The two hypothesized conformations for the g^S monomers.</p