Effects of Lys to Glu mutations in GsMTx4 on membrane binding, peptide orientation, and self-association propensity, as analyzed by molecular dynamics simulations.
GsMTx4, a gating modifier peptide acting on cationic mechanosensitive channels, has a positive charge (+5e) 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 was 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.
Teikyo University School of Medical Technology, Tokyo, Japan. Electronic address: kazunet@med.teikyo-u.ac.jp.
Kazuhisa
K
Nishizawa
Teikyo University School of Medical Technology, Tokyo, Japan.
Manami
M
Nishizawa
Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY, USA.
Radhakrishnan
R
Gnanasambandam
Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY, USA.
Frederick
F
Sachs
Department of Biology, University of Maryland, College Park, MD, USA.
Sergei I
SI
Sukharev
Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY, USA.
Thomas M
TM
Suchyna
D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013111:Spider Venoms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D036341:Intercellular Signaling Peptides and Proteins; D008051:Lipid Bilayers; C411249:MTx4 protein, Grammostola spatulata; D008563:Membrane Lipids; D010455:Peptides; D010713:Phosphatidylcholines; D013111:Spider Venoms; D018698:Glutamic Acid; D008239:Lysine; C028694:1-palmitoyl-2-oleoylphosphatidylcholine
Mechanotransduction; Membrane-active peptide; Multiscale simulations; Peptide inhibitor