Many transmembrane (TM) proteins have an amphipathic helix. These amphipathic helices can sense lipid composition, stabilize membrane curvature, and form protein-protein interactions. Here, we combined solid-state NMR spectroscopy, molecular dynamics simulations, and other biophysical techniques to characterize the structure and membrane interaction of a minimal TM and amphipathic construct (residues 101–146) from Mycobacterium tuberculosis ( Mtb ) FtsL. Amino-acid-specific 15 N-labeled oriented-sample NMR spectra in POPC:POPG (at 4:1 molar ratio) membranes uniquely defined the orientation of the TM helix (residues 124–144) including a 16° tilt but only constrained the amphipathic helix (residues 101–114) to a 90° tilt, with ambiguity in helical rotation and membrane burial depth. To resolve this ambiguity, we determined the 2-dimensional free energy surface by umbrella sampling simulations. The free energy surface contained a major minimum, with the sidechains of Leu104, Leu107, and Ile111 projected into the hydrophobic core of the membrane and those of Arg103, Arg107, and Arg114 projected sideways to interact with lipid headgroups. The structure of residues 101–114 was refined by restrained molecular dynamics simulations in a POPC:POPG bilayer. With pure POPC membranes, the free-energy barrier separating the membrane-bound state from the released state was significantly reduced (from 1.9 kcal/mol to ∼0.9 kcal/mol), showing the importance of electrostatic attraction between the Arg sidechains and the acidic POPG headgroup in keeping the amphipathic helix membrane-bound. Together, these results demonstrate that the amphipathic helix of FtsL functions as a membrane-interacting element that stabilizes the protein in the membrane environment and facilitates its incorporation into the assembling divisome for Mtb cell division.
Fan et al. (Sun,) studied this question.