Tuning of a Membrane-Perforating Antimicrobial Peptide to Selectively Target Membranes of Different Lipid Composition

Abstract

The use of designed antimicrobial peptides as drugs has been impeded by the absence of simple sequence-structure-function relationships and design rules. The likely cause is that many of these peptides permeabilize membranes via highly disordered, heterogeneous mechanisms, forming aggregates without well-defined tertiary or secondary structure. We suggest that the combination of high-throughput library screening with atomistic computer simulations can successfully address this challenge by tuning a previously developed general pore forming peptide into a selective pore former for different lipid types. A library of 2,916 peptides was designed based on the LDKA template. The library peptides were synthesized and screened using a high-throughput orthogonal vesicle leakage assay. Dyes of different sizes were entrapped inside vesicles with varying lipid composition to simultaneously screen for both pore size and affinity for negatively charged and neutral lipid membranes. From this screen, nine different LDKA variants that have unique activity were selected, sequenced, synthesized, and characterized. Despite the minor sequence changes, each of these peptides has unique functional properties, forming either small or large pores and being selective for either neutral or anionic lipid bilayers. Long-scale, unbiased atomistic molecular dynamics (MD) simulations directly reveal that rather than rigid, well-defined pores, these peptides can form a large repertoire of functional dynamic and heterogeneous aggregates, strongly affected by single mutations. Predicting the propensity to aggregate and assemble in a given environment from sequence alone holds the key to functional prediction of membrane permeabilization.

Antibacterial Activity of T22, a Specific Peptidic Ligand of the Tumoral Marker CXCR4

“…However, the distribution of charges in their surfaces is completely different, a fact that generates a much clearer difference between sides in GWH1 than in T22 (Figure 1D). The magnitudes of their hydrophobic moment (HM) vectors, a measure of amphipathicity, are in both cases high, which has been correlated with the membrane pore-formation capacity [27], a common feature of AMPs. Amphipathicity, however, is also related to toxicity over mammalian cells [105][106][107].…”

“…It has been postulated that a right balance between amphipathicity and hydrophobicity is the key to attaining a high antimicrobial activity and low toxicity [28], although it has also been described that detailed surface electrostatics [108] and the HM angle [47,109] highly influence the outcome. On the other hand, it has been proposed that an HM-vector-magnitude threshold, also modulated by the other factors mentioned, could exist that defines the onset of toxicity [27]. Thus, while T22 and GWH1 share features expected in many AMPs, such as a high HMvector magnitude and a certain tilt angle relative to the membrane normal, the slightly lower amphipathicity, higher net charge and lower hydrophobicity of T22 may result in the absence of generic toxicity and hemolysis (Figure 4B), while retaining a moderate antibacterial activity (Figure 2) and potent biofilm inhibition capacity (Figure 4).…”

“…Despite polyphemusins generically displaying potent antimicrobial activities [4,[20][21][22][23], these functionalities and their interactivity with bacterial cell membranes largely depend on the precise amino acid sequence, which is highly sensitive to even a few amino acid substitutions [24,25]. This is because even point mutations can alter its amphipathicity and hydrophobicity, features that have been postulated to be pivotal in maintaining the right balance between toxicity and antimicrobial activity [26][27][28]. Although several structural variants of polyphemusin peptides have been tested for antimicrobial properties [24,25], T22 has been never explored in this regard.…”

Membrane Association Modes of Natural Anticancer Peptides: Mechanistic Details on Helicity, Orientation, and Surface Coverage

“…The combined LD and fluorescence data suggest that while the helical peptide lies parallel to the surface of the membrane, the Trp residue points away from the helix axis towards the hydrophobic membrane interior. In contrast, for the PC-bound peptide, the rather wide fluorescence emission peak, with only a slightly blue-shifted maximum compared to the free peptide, might indicate tryptophan populations in solvent-exposed environments [88]. Tempo-La contains a single Tyr residue where the side-chain phenol group possesses two orthogonal in-plane transition moments (Figure 3B, inset).…”

A molecular dynamics study of antimicrobial peptide translocation across the outer membrane of Gram-negative bacteria

“…These studies include simulations using both coarse grained [25,26], atomistic [27][28][29][30][31] or a combination of both methods [32]. Vesicle leakage assays in combination with MD simulations have also been used to determine aggregation states and develop a molecular understanding for the AMP activity [33,34]. Additionally vesicle based experiments allow the determination of the partitioning coefficients and associated free energies of insertion [35].…”