Effects of Lys to Glu mutations in GsMTx4 on membrane binding, peptide orientation, and self-association propensity, as analyzed by molecular dynamics simulations

Abstract

GsMTx4, a gating modifier peptide acting on cationic mechanosensitive channels, has a positive charge (+5 e) due to six Lys residues. The peptide does not have a stereospecific binding site on the channel but acts from the boundary lipids within a Debye length of the pore probably by changing local stress. To gain insight into how these Lys residues interact with membranes, we performed molecular dynamics simulations of Lys to Glu mutants in parallel with our experimental work. In silico, K15E had higher affinity for 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine bilayers than wild-type (WT) peptide or any other mutant tested, and showed deeper penetration than WT, a finding consistent with the experimental data. Experimentally, the inhibitory activities of K15E and K25E were most compromised, whereas K8E and K28E inhibitory activities remained similar to WT peptide. Binding of WT in an interfacial mode did not influence membrane thickness. With interfacial binding, the direction of the dipole moments of K15E and K25E were predicted to differ from WT, whereas those of K8E and K28E oriented similarly to that of WT. These results support a model in which binding of GsMTx4 to the membrane acts like an immersible wedge that serves as a membrane expansion buffer reducing local stress and thus inhibiting channel activity. In simulations, membrane-bound WT attracted other WT peptides to form aggregates. This may account for the positive cooperativity observed in the ion channel experiments. The Lys residues seem to fine-tune the depth of membrane binding, the tilt angle, and the dipole moments.

Piezo channels and GsMTx4: Two milestones in our understanding of excitatory mechanosensitive channels and their role in pathology

“…We predicted that this creates a reservoir of mobile surface area on one monolayer which would have the effect of clamping the tension on that monolayer which was further supported by molecular dynamic simulations (Fig. 2 and [75]). A new model of GsMTx4 inhibition is proposed where the critical factors for its inhibitory potential are how deeply it binds to relaxed membranes and its availability to transition to deeper states as tension increases.…”

GsMTx4-D is a cardioprotectant against myocardial infarction during ischemia and reperfusion

“…We have shown that GsMTx4 associates superficially with membranes under low tension, and inserts deeper when membrane tension increases acting as an area/tension clamp for the outer monolayer [21,22]. Recent electron micrographic studies of the Piezo1 channel structure place the likely mechanosensing domains for these channels on the outer monolayer [59].…”

“…In vitro measures of MSC activity in cardiac tissue, single cells and patch assays, show GsMTx4 activity in the range of 0.1–5 μM [15,19,20]. GsMTx4 is a gating modifier that inhibits MSCs by clamping the membrane tension of the outer monolayer where its penetration depth, and thus its surface area contribution, is tension dependent [21,22]. …”

Molecular simulations of venom peptide‐membrane interactions: Progress and challenges

“…Independent of resolution of the force field used, classical (i.e., unbiased) MD simulation are used to address questions including, but not limited to, the spontaneous binding of the peptide to the membrane surface, including conformational changes in the peptide, the penetration‐depth and orientation of the peptide at the water–lipid interface, the identification of peptide residues that control lipid binding, the effect of peptide binding on the local structure of the membrane, and the effect of lipid composition or mutations in the peptide on these properties. For pore‐forming peptides, classical MD simulations are also used to study the structure and stability of preformed pores or the first steps in pore formation …”

“…Like GMs, MSC‐directed peptides are mostly disulfide‐rich peptides with a well‐defined secondary structure. Examples include the spider venom peptides GasFII, GsMTx‐2, and GsMTx‐4 . The third group are pore‐forming peptides that irreversibly damage cell membranes by a pore‐forming mechanism (Figure ).…”