Phosphate-Mediated Arginine Insertion into Lipid Membranes and Pore Formation by a Cationic Membrane Peptide from Solid-State NMR

Abstract

The insertion of charged amino acid residues into the hydrophobic part of lipid bilayers is energetically unfavorable yet found in many cationic membrane peptides and protein domains. To understand the mechanism of this translocation, we measured the (13)C-(31)P distances for an Arg-rich beta-hairpin antimicrobial peptide, PG-1, in the lipid membrane using solid-state NMR. Four residues, including two Arg's, scattered through the peptide were chosen for the distance measurements. Surprisingly, all residues show short distances to the lipid (31)P: 4.0-6.5 A in anionic POPE/POPG membranes and 6.5-8.0 A in zwitterionic POPC membranes. The shortest distance of 4.0 A, found for a guanidinium Czeta at the beta-turn, suggests N-H...O-P hydrogen bond formation. Torsion angle measurements of the two Arg's quantitatively confirm that the peptide adopts a beta-hairpin conformation in the lipid bilayer, and gel-phase 1H spin diffusion from water to the peptide indicates that PG-1 remains transmembrane in the gel phase of the membrane. For this transmembrane beta-hairpin peptide to have short (13)C-(31)P distances for multiple residues in the molecule, some phosphate groups must be embedded in the hydrophobic part of the membrane, with the local (31)P plane parallel to the beta-strand. This provides direct evidence for toroidal pores, where some lipid molecules change their orientation to merge the two monolayers. We propose that the driving force for this toroidal pore formation is guanidinium-phosphate complexation, where the cationic Arg residues drag the anionic phosphate groups along as they insert into the hydrophobic part of the membrane. This phosphate-mediated translocation of guanidinium ions may underlie the activity of other Arg-rich antimocrobial peptides and may be common among cationic membrane proteins.

Molecular dynamics simulations suggest a mechanism for translocation of the HIV-1 TAT peptide across lipid membranes

“…In the translocation of the Tat peptide, we find that the membrane transiently changes its local topology by forming a pore that facilitates the diffusion of the peptide on its surface, allowing it to reach the distal side. This level of membrane flexibility and the ubiquitous nature of the phosphate-guanidinium interactions may be essential in several other biological mechanisms, such as pore formation by antimicrobial peptides (39,42), voltage-gated ion channels, and flippase regulation of lipid distribution on different faces of a bilayer (43,44).…”

Structure and dynamics of cationic membrane peptides and proteins: Insights from solid‐state NMR

“…51 For the first time, short Arg Cf-P distances (4.0-5.7 Å ) were found, indicating H-bonding of the guanidinium with the phosphates. This short distance was true even for an Arg residue in the middle of the TM b-strand.…”

“…1(f)]. 51 Water-protein H-bonding, which may be involved in charge solvation, can be detected using 2D 13 C-1 H and 15 N-1 H correlation experiments. 52 …”

Interactions between the Conserved Hydrophobic Region of the Prion Protein and Dodecylphosphocholine Micelles

“…Therefore, the asymmetry suggests the presence of stabilizing electrostatic interactions, perhaps of the salt bridge type, involving the guanidinium group of Arg 136 and the phosphate moiety of the detergent headgroup. Salt bridge interactions have been observed in membrane-bound cell-penetrating peptides such as penetratin and HIV-TAT (34,35).…”