Abstract
Phosphoinositides are essential signaling lipids that play a critical role in regulating ion channels, and their dysregulation often results in fatal diseases including cardiac arrhythmia and paralysis. Despite decades of intensive research, the underlying molecular mechanism of lipid agonism and specificity remains largely unknown. Here, we present a systematic study of the binding mechanism and specificity of a native agonist, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and two of its variants, PI(3,4)P2 and PI(3,4,5)P3, on inwardly rectifying potassium channel Kir2.2, using molecular dynamics simulations and free energy perturbations (FEPs). Our results demonstrate that the major driving force for the PI(4,5)P2 specificity on Kir2.2 comes from the highly organized salt-bridge network formed between the charged inositol head and phosphodiester linker of PI(4,5)P2. The unsaturated arachidonic chain is also shown to contribute to the stable binding through hydrophobic interactions with nearby Kir2.2 hydrophobic residues. Consistent with previous experimental findings, our FEP results confirmed that non-native ligands, PI(3,4)P2 and PI(3,4,5)P3, show significant loss in binding affinity as a result of the substantial shift from the native binding mode and unfavorable local solvation environment. However, surprisingly, the underlying molecular pictures for the unfavorable binding of both ligands are quite distinctive: for PI(3,4)P2, it is due to a direct destabilization in the bound state, whereas for PI(3,4,5)P3, it is due to a relative stabilization in its free state. Our findings not only provide a theoretical basis for the ligand specificity, but also generate new insights into the allosteric modulation of ligand-gated ion channels.
Highlights
The most abundant phosphoinositide in membranes is phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) whose well-known functional roles include the generation of two critical second messengers, inositol trisphosphate (IP3) and diacyl glycerol (DAG) a er being hydrolyzed, as well as its broad regulation of almost all ion channels and many transporters.[7,8,9,10,11]
The free energy perturbations (FEPs) calculation shows a 13.9 kcalmolÀ1 (DDG) and 39.7 kcalmolÀ1 for PI(3,4,5)P3 and PI(3,4)P2, respectively, indicating that the variants are less speci c to the Kir2.2 channel and con rming the experimental ndings on the mouse Kir2.2.21 Our results reveal that the mutation shi ed the binding modes from what the native ligand possessed, which was accompanied by the unfavorable solvation environment change around the ligands which eventually affected the channel gating efficiency
The channel complex as well as each subunit remains stable through the entire simulation, as indicated in the stable root mean square deviation (RMSD) uctuation and consistent root mean square uctuation (RMSF) patterns (Fig. S1†).The overall binding mode is summarized in Fig. 2a and b
Summary
Phosphoinositides (PIPs) are one class of signaling lipid molecules distributed broadly in the inner lea et of the plasma membrane, which play critical roles in diverse physiological functions, such as regulating the activity of ion channels and transporters, endocytosis and exocytosis, and calcium signaling.[1,2,3,4,5,6] The most abundant phosphoinositide in membranes is phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) whose well-known functional roles include the generation of two critical second messengers, inositol trisphosphate (IP3) and diacyl glycerol (DAG) a er being hydrolyzed, as well as its broad regulation of almost all ion channels and many transporters.[7,8,9,10,11] Structurally, PI(4,5)P2 is composed of an inositol head group with two phosphates on its 40. Regarding the electrostatic interaction for the PI(4,5)P2–Kir channel, it is interesting to raise the question whether Kir could be activated by other phosphoinositides like PI(3,4)P2 and PI(3,4,5)P3, which have high structural and chemical similarity to the native agonist PI(4,5)P2 Both PI(3,4)P2 and PI(3,4,5)P3 are phospholipid components of cell membranes involved in many important signal transduction pathways. Logothetis's lab recently utilized electrophysiology techniques to measure the activity of a series of Kir channels, Kir2.1, Kir2.2, K3.4*, and Kir6.2, repetitively, under PI(4,5)P2, PI(3,4)P2 and PI(3,4,5)P3.21 They discovered that on one extreme PI(3,4)P2 and PI(3,4,5)P3 hardly activate the channel (i.e., Kir2.1, a typical Kir channel that requires PI(4,5)P2 for maintaining normal function) (Group 1); and on the other extreme, the current levels induced by PI(4,5)P2, PI(3,4,5)P3, or PI(3,4)P2 were comparable (i.e., Kir6.2, sensitive to long chain acyl CoA) (Group 4) In other cases, their responses to PI(3,4)P2 and PI(3,4,5)P3 were in-between Kir2.1 and Kir6.2. The FEP calculation shows a 13.9 kcalmolÀ1 (DDG) and 39.7 kcalmolÀ1 for PI(3,4,5)P3 and PI(3,4)P2, respectively, indicating that the variants are less speci c to the Kir2.2 channel and con rming the experimental ndings on the mouse Kir2.2.21 Our results reveal that the mutation shi ed the binding modes from what the native ligand possessed, which was accompanied by the unfavorable solvation environment change around the ligands which eventually affected the channel gating efficiency
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