Structural and Functional Mimicry of the Antimicrobial Defensin Plectasin by Analogues with Engineered Backbone Composition

Abstract

The threat posed by bacteria resistant to common antibiotics creates an urgent need for novel antimicrobials. Non-ribosomal peptide natural products that bind Lipid II, such as vancomycin, represent a promising source for such agents. The fungal defensin plectasin is one of a family of ribosomally produced miniproteins that also exert antimicrobial activity via Lipid II binding. Made up entirely of canonical amino acids, these molecules are potentially more susceptible to degradation by protease enzymes than non-ribosomal counterparts. Here, we report the development of proteomimetic variants of plectasin through the systematic incorporation of artificial backbone connectivity in the domain. An iterative secondary-structure-based design scheme yields a variant with a tertiary fold indistinguishable from the prototype natural product, potent activity against Gram positive bacteria, and low mammalian cell toxicity. Backbone modification is shown to improve oxidative folding efficiency of the disulfide-rich scaffold as well as resistance to proteolytic hydrolysis. These results broaden the scope of design strategies toward protein mimetics as well as folds and biological functions possible in such agents.

Keywords: antimicrobial peptide; disulfide-rich peptide; heterogeneous backbone; plectasin; proteomimetic.

Figure 1

(A) Sequence and secondary structure map of plec, NZ2114, and heterogeneous‐backbone variants. Residues in bold are substitutions relative to NZ2114. “R” groups, when present but not specified, match side chain of the α‐residue indicated by the single letter code in the sequence. (B) HPLC chromatograms for oxidative folding reactions (normalized to total peak area). The highlighted peak corresponds to the fully oxidized product, and crude purity is shown in parentheses. Reactions consisted of 0.5 mg/mL reduced linear precursor in 100 mM Tris, 1 M guanidinium chloride, 7.7 mM glutathione, 1.0 mM oxidized glutathione at pH 8.5 and were analyzed after 3.5 h.

Figure 2

X‐ray crystal structure of plec (PDB 3E7U) and NMR structure ensembles (10 models per sequence) for NZ2114 and variants. Positions of backbone modification are indicated by spheres and colored by residue type. Backbone rmsd (average and standard deviation) for variant ensemble overlay with the plec X‐ray structure is shown in parentheses.

Figure 3

Cytotoxicity of NZ2114, heterogeneous‐backbone variants, and human cathelicidin LL‐37 (LL37). Graphs depict concentration‐dependent effect on cell viability of the indicated cell line 24 h after treatment.

Figure 4

Comparison of proteolytic stability of NZ2114 and var4. (A) Fraction of intact full‐length peptide remaining as a function of time for each sequence in the presence of trypsin (36 μM enzyme in 100 mM NH₄HCO₃, 1 mM CaCl₂, pH 8.5) or pronase (0.2 mg/mL enzyme in 100 mM NH₄OAc, 1 mM CaCl₂, pH=7.2). (B) Hydrolysis sites identified by MS analysis of quenched reaction mixtures after reduction of disulfides with tris‐carboxyethyl phosphine.