Pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission and are sensitive to their lipid environment. The mechanism of phospholipid modulation of any pLGIC is not well understood. We demonstrate that the model pLGIC, ELIC (Erwinia ligand-gated ion channel), is positively modulated by the anionic phospholipid, phosphatidylglycerol, from the outer leaflet of the membrane. To explore the mechanism of phosphatidylglycerol modulation, we determine a structure of ELIC in an open-channel conformation. The structure shows a bound phospholipid in an outer leaflet site, and structural changes in the phospholipid binding site unique to the open-channel. In combination with streamlined alchemical free energy perturbation calculations and functional measurements in asymmetric liposomes, the data support a mechanism by which an anionic phospholipid stabilizes the activated, open-channel state of a pLGIC by specific, state-dependent binding to this site.
Free Energy Perturbation (FEP) is a powerful but challenging computational technique for estimating differences in free energy between two or more states. This document is intended both as a tutorial and as an adaptable protocol for computing free energies of binding using free energy perturbations in NAMD. We present the Streamlined Alchemical Free Energy Perturbation (SAFEP) framework. SAFEP shifts the computational frame of reference from the ligand to the binding site itself. This both simplifies the thermodynamic cycle and makes the approach more broadly applicable to superficial sites and other less common geometries. As a practical example, we give instructions for calculating the absolute binding free energy of phenol to lysozyme. We assume familiarity with standard procedures for setting up, running, and analyzing molecular dynamics simulations using NAMD and VMD. While simulation times will vary, the human tasks should take no more than 3 to 4 hours for a reader without previous training in free energy calculations or experience with the Colvars Dashboard. Sample data are provided for all key calculations both for comparison and readers' convenience.
Pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission and are sensitive to their lipid environment. The mechanism of phospholipid modulation of any pLGIC is not well understood. We demonstrate that the model pLGIC, ELIC (Erwinia ligand-gated ion channel), is positively modulated by the anionic phospholipid, phosphatidylglycerol, from the outer leaflet of the membrane. To elucidate the mechanism of phosphatidylglycerol modulation, we determine a structure of ELIC in an open conformation. The structure shows a bound phospholipid in an outer leaflet site, and conformational changes in the phospholipid binding site unique to the open state. In combination with streamlined alchemical free energy perturbation calculations and functional measurements in asymmetric liposomes, the data support a mechanism by which an anionic phospholipid stabilizes the open state of a pLGIC by specific, state-dependent binding to this site.
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