Punnathanam, S. | Search bioRxiv

bioRxiv2026

Cardiolipin regulates free energy of folding and vesicle leakage of CM15 antimicrobial peptides in bacterial inner membranes

Globally rising antimicrobial resistance has led to antimicrobial peptides (AMPs) being explored as alternative therapeutics for bacterial infections. A better mechanistic understanding of the interaction of the peptide with membrane compositions can aid in targeting specific bacterial strains more effectively and lead to more resilient treatments. Using all-atom molecular dynamics simulations, we obtain the free-energy landscape of the cecropin-melittin hybrid peptide CM15 using a path-based method and investigate the effect of cardiolipin (CL), a four - tailed lipid found in bacterial membranes. Our analysis reveals that the transition of CM15 from an unfolded state in solution to a membrane-bound helical state proceeds through three main steps: membrane binding, insertion in an unfolded state, and subsequent folding beneath the lipid headgroups into an -helical conformation. In a phosphatidylethanolamine (PE)/phosphatidylglycerol (PG) membrane lacking CL, folding occurs spontaneously without appreciable free energy barriers. However, the addition of only 5% CL substantially alters the landscape, giving rise to two distinct transition pathways with barriers ranging from 2.5 to 8 k BT. This reflects the presence of a more rugged free-energy landscape associated with local heterogeneity in lipid composition. The emergence of these barriers is driven by strong interactions between CL and CM15, which promote CL sequestration around the peptide in regions of negative membrane curvature. Vesicle leakage experiments reveal the inhibitory influence of CL at lower peptide-to-lipid ratios with delayed kinetics as the CL content is increased. We attribute the reduced activity at low CM15 concentrations to the free-energy barriers associated with peptide folding and to strong CM15-CL interactions that stabilize a membrane bound peptide state, thereby hindering membrane disruption, pore formation, and leakage. Our study illustrates the putative role of CL in pore formation, providing molecular insights that can potentially aid the rational design of strain-specific synthetic AMPs.

biophysics↗

bioRxiv2021

Conformational flexibility is a key determinant of the lytic activity of the pore forming protein, Cytolysin A

Bacterial pore-forming toxins (PFTs) bind and oligomerize on mammalian cell membranes forming nanopores, that cause cell lysis to promote a wide range of bacterial infections. Cytolysin A (ClyA), an alpha()-PFT, is known to undergo one of the largest conformational changes during its transition from a water soluble monomeric form to the membrane embedded dodecameric nanopore assembly. Despite extensive work on the structure and assembly of ClyA, a complete molecular picture of the interplay between the protein segments and membrane lipids driving this transformation remains elusive. In this study, we combine experiments and all-atom molecular dynamics (MD) simulations of ClyA and its mutants to unravel the role of the two key membrane interacting motifs, namely, the {beta}-tongue and N-terminus helix, in facilitating this critical transition. Erythrocyte turbidity and vesicle leakage assays reveal a loss of activity for {beta}-tongue mutant (Y178F), and delayed kinetics for the N-terminus mutants (Y27A and Y27F). All atom, thermal unfolding molecular dynamics simulations of the monomer carried out at 310, 350 and 400 K reveal a distinct reduction in the flexibility in both the {beta}-tongue and N-terminal regions of the mutants when compared with the wild type. This decreased loss of conformational flexbility correlates positively with the reduced lytic and leakage activity observed in experiments, indicating that the tendency to lose secondary structure in the {beta}-tongue region is an important step in the conformational transition bistability of the ClyA protein. Simulations with the membrane inserted oligomeric arcs representing the pore state reveal a greater destabilization tendency among the {beta}-tongue mutant as inferred from secondary structure and N-terminal positioning. Our combined experimental and simulation study, reveals that conformational flexibility is indispensable for the outward movement of the {beta}-tongue and the tendency to induce disorder in the {beta}-tongue is an important step in the transition to the membrane mediated helix-turn-helix motif integral to ClyA pore formation. This observed loss of secondary structure is akin to the structural transitions observed in intrinsically disordered proteins (IDPs) to support protein function. Our finding suggest that inherent flexibility in the protein could play a wider and hitherto unrecognized role in the membrane mediated conformational transitions of PFTs in general. Author summaryBacterial pore-forming toxins (PFTs) bind and oligomerize on mammalian cell membranes to form bilayer spanning nanopores. Unregulated pore formation disrupts ionic balance that compromises the permeability of the cell, leading to cell death and infection. PFTs display remarkable structural plasticity that allow these proteins to interconvert between the two physiochemically distinct water soluble and membrane bound forms. However the molecular mechanism for this robust interconversion is poorly understood. For example, upon membrane binding, cytolysin A (ClyA), an -PFT, shows one of the largest conformational changes in protein structure among the PFT family of proteins. In order to understand this transtition we characterized several point mutations in ClyA using experiments and molecular dynamics (MD) simulations to understand the role of two essential ClyA motifs ({beta} tongue and N-terminus) implicated in the conformational changes responsible for oligomerization and conferring stability to the pore state. Our study reveals that the innate conformational flexibility of the {beta} tongue results in a disordered intermediate state that facilitate the complete transition to the pore state. This tendency to disorder is compromised to varying degrees in ClyA mutants, correlating with the loss of lytic activity. Our results suggest that the finely tuned conformational flexibility in the membrane motifs of ClyA are critical to its function, revealing a broader paradigm that could be at play during membrane associated secondary structure transitions of proteins in general.

biophysics↗