Direct binding of phosphatidylglycerol at specific sites modulates desensitization of a 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, a mutation that decreases 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

Dynamics of Aromatic Side Chains in the Active Site of FKBP12

FKBP12, a small human enzyme, aids protein folding by catalyzing cis-trans isomerization of peptidyl-prolyl bonds, and is involved in cell signaling pathways, calcium regulation, and the immune response. The underlying molecular mechanisms are not fully understood, but it is well-known that aromatic residues in the active site and neighboring loops are important for substrate binding and catalysis. Here we report micro- to millisecond exchange dynamics of aromatic side chains in the active site region of ligand-free FKBP12, involving a minor state population of 0.5% and an exchange rate of 3600 s-1, similar to previous results for the backbone and methyl-bearing side chains. The exchange process involves tautomerization of H87. In the major state H87 is highly flexible and occupies the common HNε2 tautomer, while in the minor state it occupies the rare HNδ1 tautomer, which typically requires stabilization by specific interactions, such as hydrogen bonds. This finding suggests that the exchange process is coupled to a rearrangement of the hydrogen bond network around H87. Upon addition of the active-site inhibitor FK506 the exchange of all aromatic residues is quenched, with exception of H87. The H87 resonances are broadened beyond detection, suggesting that interconversion between tautomers prevail in the FK506-bound state. While key active-site residues undergo conformational exchange in the apo state, the exchange rate is considerably faster than the catalytic turnover, as determined herein by Michaelis-Menten type analysis of NMR line shapes and chemical shifts. We discuss alternative interpretations of this observation in terms of FKBP12 function