Effect of targeted minimal sequence variations on the structure and biological activities of the human cathelicidin LL‐37

Abstract

LL‐37 is an innate immune peptide derived from the human cathelicidin, which exerts pleiotropic roles in host defense and healing. These activities in part depend on its capacity to adopt an amphipathic helical structure in physiological solutions and then oligomerizing. Orthologues from other primates, such as rhesus RL‐37, remain monomeric and disordered under the same conditions. Intramolecular salt‐bridges, arising from appropriately spaced anionic and cationic residues in its sequence, may play a relevant role in determining the particular structure adopted by LL‐37. To probe this, we have effected minimal, targeted residue variations such as replacement of a single residue (K15→G), or inversion of one or both sets of two residues (E10 K11→ K10 E11 or E16 K18→ K16 E18). This could alter the pattern of intramolecular salt bridging without affecting other functionally relevant parameters such as overall hydrophobicity, helix amphipathicity or charge. The structural and functional effects were analyzed using CD spectroscopy, surface plasmon resonance, antimicrobial activity assays, and bacterial membrane permeabilization to fluorescent probes of increasing sizes, using flow cytometry. Analogs were functionally different from both LL‐37 and RL‐37, so it was not possible to switch from the function of one to that of the other simply by altering the salt‐bridging pattern in this manner. This indicates that the particular structure/function characteristics of LL‐37 likely depend quite subtly, and in a precise and complex manner, on a complex pattern of intramolecular interactions.

Relating Molecular Dynamics Simulations to Functional Activity for Gly-Rich Membranolytic Helical Kiadin Peptides

“…Kiadin-2, with a N-terminal Val 5 →Gly substitution, binds with a similar affinity to diPGLa-H up to a peptide concentration of 8 µM (Figure 8). However, at 16 µM, after an initial increase in the response, there is a sharp drop in the RU, a behaviour we have previously associated with an AMP-induced LUV breakup [19]. Kiadin-6, which also has a single substitution (Val 14 →Gly), shows the strongest increase in the RU for peptide binding (Figure 8), suggesting a higher affinity for neutral LUVs, consistent with CD observations.…”

“…In this respect, interactions with components of the extracellular medium and of the bacterial outer membrane and/or peptidoglycan layers can be as important in defining their potency and spectrum of activity as the lipid compositions of the bacterial membranes they eventually interact with and disrupt [ 16 , 17 , 18 ]. Finally, the tendency of the peptides to remain monomeric or to oligomerize also significantly affects their mode of action and potency [ 19 ]. Considering that all these features must be correctly balanced to enhance the antibacterial potency while, at the same time, limiting the toxicity to host cells, it is understandable that AMPs must have quite subtle structure–activity relationship (SAR) characteristics.…”

Design, antimicrobial activity and mechanism of action of Arg-rich ultra-short cationic lipopeptides

“…This increase was more pronounced for Lp-I in agreement with the larger size of aggregates formed by this molecule than Lp-I RR (see S2 Fig ). Furthermore, it is worth to note that only for Lp-I, the shape of this sensorgram changes, with a loss of RU during the injection, suggesting a putative disaggregation of membrane-bound aggregates, without disruptive effect of the LUVs [40].…”

Antimicrobial Peptides as Anti-Infective Agents in Pre-Post-Antibiotic Era?

“…In fact, the same consideration could be made for other components of the microbial cell wall, but the data is less consistent. However, even limiting one’s focus to the lipid bilayer, there is still an imperfect understanding of the complex relationship between the AMP and membrane, since even peptides with very similar structures can have remarkably different reported mechanisms of action (e.g., buforin and magainin 2, or LL-37 and the rhesus orthologue RL-37) [71,72,73]. A better understanding of the relationship between physico-chemical properties and biological activity is, therefore, required to identify features that are responsible for potency and specificity of AMPs.…”

“…In principle, the distribution of positively charged residues should not correlate with peptide potency, which should only depend on the overall peptide charge [81,104]. However, it can make a significant difference if it affects the formation of helix-stabilizing salt bridges, as observed in artificial variants of the human helical peptide LL-37 [73].…”