The limits of prediction: Why intrinsically disordered regions challenge our understanding of antimicrobial peptides

Abstract

Antimicrobial peptides (AMPs) are molecules found in most organisms, playing a vital role in innate immune defense against pathogens. Their mechanism of action involves the disruption of bacterial cell membranes, causing leakage of cellular contents and ultimately leading to cell death. While AMPs typically lack a defined structure in solution, they often assume a defined conformation when interacting with bacterial membranes. Given this structural flexibility, we investigated whether intrinsically disordered regions (IDRs) with AMP-like properties could exhibit antimicrobial activity. We tested 14 peptides from different IDRs predicted to have antimicrobial activity and found that nearly all of them did not display the anticipated effects. These peptides failed to adopt a defined secondary structure and had compromised membrane interactions, resulting in a lack of antimicrobial activity. We hypothesize that evolutionary constraints may prevent IDRs from folding, even in membrane-like environments, limiting their antimicrobial potential. Moreover, our research reveals that current antimicrobial predictors fail to accurately capture the structural features of peptides when dealing with intrinsically unstructured sequences. Hence, the results presented here may have far-reaching implications for designing and improving antimicrobial strategies and therapies against infectious diseases.

Keywords: Antimicrobial peptide; Disordered proteins; Peptide design; Prediction.

Fig. 1

Analysis of disordered proteins and amino acid propensities. (A) Proportional comparison of amino acid types in canonical AMPs versus IDR-encrypted AMPs, (B) Difference in amino acid percentages in canonical AMPs compared to IDR-encrypted AMPs, (C) Correlation between amino acids' antimicrobial and folding propensities. Amino acid propensities for order and disorder, based on the DisProt scale , are depicted as circles and diamonds, respectively. Amino acids with predicted antimicrobial propensities, as determined by M. Torrent et al. , are highlighted in red (low propensity) and green (high propensity), (D) Representation of ordered and disordered regions in various tissues through pie charts, (E) Tissue-specific disordered fraction predictions using the espN tool . Error bars correspond to the standard error of the mean. The ANOVA test was used to evaluate significant differences between the brain and other tissues (denoted as * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001), (F) Diagram showing neurotrophin-3's disorder (red) and antimicrobial (green) predictions with threshold markers (0.5 and 0.225, respectively) in dashed lines. The selected peptide of neurotrophin-3 (purple segment) is from the segment with highest antimicrobial propensity.

Fig. 2

Structural characteristics of control peptides. The figure presents the structural properties of control peptides H1 (A), H2 (B), and H3 (C), showing various aspects across different panels. The left panel shows the CD profile in water (black) and SDS micelles (red), alongside the structure in the original protein (purple), PepFold prediction (orange), and AlphaFold prediction (blue). In the right panel, membrane interactions are measured through the intrinsic fluorescence of tyrosine (H1 and H3) and tryptophan (H2), showing a marked decrease in fluorescence upon membrane binding. Additionally, the helical peptide regions in contact with or embedded in the membrane are displayed, as predicted by the FMAP model.

Fig. 3

Structural Characteristics of Peptides 5, 9 and, 10. The figure presents the structural properties of control peptides 5 (A), 9 (B), and 10 (C), showing various aspects across different panels. The left panel shows the CD profile in water (black) and SDS micelles (red), alongside the structure in the original protein (purple), PepFold prediction (orange), and AlphaFold prediction (blue). In the right panel, membrane interactions are measured through the intrinsic fluorescence of tyrosine (5) and tryptophan (9 and 10), showing the decrease in fluorescence upon membrane binding. Additionally, the helical peptide regions in contact with or embedded in the membrane are displayed, as predicted by the FMAP model.