Effect of Leu/Val Mutation on the Energetics of Antimicrobial Peptide:Micelle Binding

Recently, we had reported a synthetic positively charged
leucine-rich
14-residue-long antimicrobial peptide (AMP, LL-14: NH3+-LKWLKKLLKWLKKL-CONH2), which was highly active
and cytotoxic relative to its valine analogue (VV-14). However, the
thermodynamics underlying this differential toxicity and antimicrobial
activity was unclear. Understanding the energetics of peptide binding
to micelles (simplest membrane mimic, viz., SDS as a bacterial membrane
and DPC as a eukaryotic membrane) and the effect of Leu → Val
peptide mutations on the stability of the peptide:micelle complexes
are of great academic interest and relevant for the rational design
of potent and selective AMPs for therapeutic use. Here, we have reported
the molecular dynamics free energy simulations that allowed us to
quantitatively estimate the strength of peptide discrimination (based
on single- or multiple-site Leu/Val mutations in LL-14) by membrane
mimetic micelles (SDS and DPC) and decipher the energetics underlying
peptide selectivity by micelles. The Leu-containing peptide (LL-14)
was found to be preferred for micelle (SDS and DPC) binding relative
to its Val analogues (single or multiple Val mutants). The strength
of the preference depended on the position of the Leu/Val mutation
in the peptide. Surprisingly, the N-terminal LL-14 single mutation
(Leu → Val: L1V) was found to fine-tune the electrostatic interactions,
resulting in the highest peptide selectivity (ΔΔG ∼ 8 kcal/mol for both SDS and DPC). However, the
mechanism of L1V peptide selectivity was distinctly different for
SDS and DPC micelles. SDS ensured high selectivity by disrupting the
peptide:micelle salt bridge, whereas DPC desolvated the broken-peptide-backbone
hydrogen bond in the V1 peptide:micelle complex. Mutations (Leu →
Val) in the middle positions of the LL-14 (4th, 7th, 8th, and 11th)
were disfavored by the micelles primarily due to the loss of peptide:micelle
hydrophobic interactions. Peptides differing at the C-terminal (i.e.,
L14V) were recognized by SDS micelles (ΔΔG ∼ 4 kcal/mol) by altering peptide:micelle interactions. L14V
mutation, on the other hand, did not play any role in the peptide:DPC
binding, as no direct interactions between the C-terminal and DPC
micelle were observed due to obvious electrostatic reasons. The strength
of selectivity favoring LL-14 binding against VV-14 was found to be
much higher for DPC micelles (ΔΔG ∼
25 kcal/mol) relative to SDS micelles (ΔΔG ∼ 19 kcal/mol). The loss of the peptide:micelle hydrophobic
contact in response to LL-14 → VV-14 mutation was found to
be significantly larger for DPC relative to SDS micelles, resulting
in higher discriminatory power for the former. Peptide:SDS salt bridges
seemed to prevent the loss of peptide:micelle hydrophobic contact
to some extent, leading to weaker selectivity for SDS micelles. High
selectivity of DPC micelles provided an efficient mechanism for VV-14
dissociation from DPC micelles, whereas low-selectivity of SDS micelles
ensured binding of both LL-14 and VV-14. To the best of our knowledge,
this is the first study in which the experimental observations (antimicrobial
activity and toxicity) between leucine-rich and valine-rich peptides
have been explained by establishing a direct link between the energetics
and structures.