Biology subjects

Petroff, J. T.

Publications and source records attributed to Petroff, J. T..

4 recordsLinked to original sources

bioRxiv2022

Lipid nanodisc scaffold and size alters the structure of a pentameric ligand-gated ion channel

Lipid nanodiscs have become the standard reconstitution system for structural and biochemical studies of membrane proteins, especially using single particle cryo-EM. We find that reconstitution of the pentameric ligand-gated ion channel (pLGIC), Erwinia ligand-gated ion channel (ELIC), in different nanodisc scaffolds (MSP1E3D1, SMA, saposin, spMSP1D1) produces distinct apo and agonist-bound structures. In the presence of agonist, different nanodiscs scaffolds produce concerted conformational changes associated with activation in ELIC, with larger nanodiscs showing more activated conformations. The effect of different nanodisc scaffolds on ELIC structure extends to the extracellular domain and agonist binding site. Molecular dynamic simulations of ELIC in small and large nanodiscs suggest that the impact of the nanodisc on ELIC structure is influenced by nanodisc size. Overall, the results indicate that the nanodisc profoundly affects the structure of a pLGIC, and suggest that larger circularized nanodiscs may be advantageous to approximate a lipid membrane environment.

biophysics↗

bioRxiv2022

Structural mechanism of leaflet-specific phospholipid modulation of a pentameric ligand-gated ion channel

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.

biophysics↗

bioRxiv2021

Polyunsaturated fatty acids inhibit a pentameric ligand-gated ion channel through one of two binding sites

Polyunsaturated fatty acids (PUFAs) inhibit pentameric ligand-gated ion channels (pLGICs) but the mechanism of inhibition is not well understood. The PUFA, docosahexaenoic acid (DHA), inhibits agonist responses of the pLGIC, ELIC, more effectively than palmitic acid, similar to the effects observed in the GABAA receptor and nicotinic acetylcholine receptor. Using photo-affinity labeling and coarse-grained molecular dynamics simulations, we identified two fatty acid binding sites in the outer transmembrane domain (TMD) of ELIC. Fatty acid binding to the photolabeled sites is selective for DHA over palmitic acid, and specific for an agonist-bound state. Hexadecyl-methanethiosulfonate modification of one of the two fatty acid binding sites in the outer TMD recapitulates the inhibitory effect of PUFAs in ELIC. The results demonstrate that DHA selectively binds to multiple sites in the outer TMD of ELIC, but that state-dependent binding to a single intrasubunit site mediates DHA inhibition of ELIC.

biochemistry↗

bioRxiv2019

Direct Binding of Phosphatidylglycerol at Specific Sites Modulates Desensitization of a Pentameric Ligand-Gated Ion Channel

Pentameric ligand-gated ion channels (pLGICs) are essential determinants of synaptic transmission, and are modulated by specific lipids including anionic phospholipids. The exact modulatory effect of anionic phospholipids in pLGICs and the mechanism of this effect are not well understood. Using native mass spectrometry, coarse-grained molecular dynamics simulations and functional assays, we show that the anionic phospholipid, 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), preferentially binds to and stabilizes the pLGIC, Erwinia ligand-gated ion channel (ELIC), and decreases ELIC desensitization. Mutations of five arginines located in the interfacial regions of the transmembrane domain (TMD) reduce POPG binding, and a subset of these mutations increase ELIC desensitization. In contrast, the L240A mutant known to decrease ELIC desensitization, increases POPG binding. The results support a mechanism by which POPG stabilizes the open state of ELIC relative to the desensitized state by direct binding at specific sites.

biophysics↗