Structural characterization of two pore‐forming peptides: Consequences of introducing a C‐terminal tryptophan

Abstract

Synthetic channel-forming peptides that can restore chloride conductance across epithelial membranes could provide a novel treatment of channelopathies such as cystic fibrosis. Among series of 22-residue peptides derived from the second transmembrane segment of the glycine receptor α 1 -subunit (M2GlyR), p22-S22W (KKKKP ARVGL GITTV LTMTT QW) is particularly promising with robust membrane insertion and assembly. The concentration to reach one-half maximal short circuit current is reduced to 45±6 μM from that of 210±70 μM of peptide p22 (KKKKP ARVGL GITTV LTMTT QS). However, this is accompanied with nearly 50% reduction in conductance. Towards obtaining a molecular level understanding of the channel activities, we combine information from solution NMR, existing biophysical data, and molecular modeling to construct atomistic models of the putative pentameric channels of p22 and p22-S22W. Simulations in membrane bilayers demonstrate that these structural models, even though highly flexible, are stable and remain adequately open for ion conductance. The membraneanchoring tryptophan residues not only rigidify the whole channel, suggesting increased stability, but also lead to global changes in the pore profile. Specifically, the p22-S22W pore has a smaller opening on average, consistent with lower measured conductance. Direct observation of several incidences of chloride transport suggests several qualitative features of how these channels might selectively conduct anions. The current study thus helps to rationalize the functional consequences of introducing a single C-terminal tryptophan. Availability of these structural models also paves the way for future work to rationally modify and improve M2GlyR-derived peptides toward potential peptide-based channel replacement therapy.

Structural and biophysical properties of a synthetic channel-forming peptide: Designing a clinically relevant anion selective pore

“…The length of production simulation was 100 ns for left-handed and straight pore assemblies, and extended to 200 ns for the right-handed model to probe the preferred handedness of the pore assembly further. These simulations are much longer than those published previously for NK 4 -M2GlyR p22 and NK 4 -M2GlyR p22-S22W pores [31]. The purpose of longer simulations is to better exploit the force field to resolve the handedness of pore assembly, a key feature that has not been determined experimentally.…”

“…Three models of the putative pentameric pore were constructed with three different handedness orientations of helix packing: left-handed, straight and right-handed, using a strategy similar to previous work [31]. The pentameric structures were solvated in pre-equilibrated POPC bilayers using the CHARMM-GUI automated Membrane Builder [40, 41].…”

“…The box size was ~65 × 65 × 72 Å. A protocol similar to one used in previous work [31] was applied to equilibrate the system at 303 K using CHARMM [42, 43], during which various restraints were applied to selected heavy atoms with gradually decreasing restraint strength. SHAKE was used to fix the length of hydrogen-attaching bonds [44], particle mesh Ewald (PME) [45] was used for long-range electrostatic interactions, and van der Waals interactions were smoothly switched off from 10 to 12 Å.…”

“…Tryptophan in proteins with transmembrane segments is known for its specific affinity for sites near the lipid carbonyl region [25]. A tryptophan containing sequence exhibited a decrease in the concentration required for net transepithelial ion conductance and an increase in the ability to adopt a helical secondary structure [29–31]. Furthermore, tryptophan and arginine reportedly play roles as anchors of transmembrane helices [32].…”

Fluorescence spectroscopy and molecular dynamics simulations in studies on the mechanism of membrane destabilization by antimicrobial peptides

“…In this way the authors proposed a structure in terms of a regular pore comprising six peptides, in agreement with the conductance levels measured in experiments with planar bilayers. Even though this approach has now been applied to other peptides [132,133] it always needs to be based on one or more competing hypotheses of the pore structure, and therefore it is better suited to molecules supposed to form ordered transmembrane barrel-stave pores. Insertion of peptides into a preequilibrated bilayer can take place only after a proper removal of lipids to make space for the peptides, either manually or driving them out with a weak repulsive potential.…”