RESUMO
The pleckstrin homology (PH) domain of the general receptor for phosphoinositides 1 (GRP1) exhibits specific, high-affinity, reversible binding to phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P(3)) at the plasma membrane, but the nature and extent of the interaction between this bound complex and the surrounding membrane environment remains unclear. Combining equilibrium and nonequilibrium molecular dynamics (MD) simulations, NMR spectroscopy, and monolayer penetration experiments, we characterize the membrane-associated state of GRP1-PH. MD simulations show loops flanking the binding site supplement the interaction with PI(3,4,5)P(3) through multiple contacts with the lipid bilayer. NMR data show large perturbations in chemical shift for these loop regions on binding to PI(3,4,5)P(3)-containing DPC micelles. Monolayer penetration experiments and further MD simulations demonstrate that mutating hydrophobic residues to polar residues in the flanking loops reduces membrane penetration. This supports a "dual-recognition" model of binding, with specific GRP1-PH-PI(3,4,5)P(3) interactions supplemented by interactions of loop regions with the lipid bilayer.
Assuntos
Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Fosfolipídeos/química , Receptores Citoplasmáticos e Nucleares/química , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Membranas Artificiais , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Fosfatos de Fosfatidilinositol/química , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/genética , Homologia Estrutural de ProteínaRESUMO
Two-pore domain potassium (K(2P)) channels play a key role in setting the membrane potential of excitable cells. Despite their role as putative targets for drugs and general anesthetics, little is known about the structure and the drug binding site of K(2P) channels. We describe A1899 as a potent and highly selective blocker of the K(2P) channel TASK-1. As A1899 acts as an open-channel blocker and binds to residues forming the wall of the central cavity, the drug was used to further our understanding of the channel pore. Using alanine mutagenesis screens, we have identified residues in both pore loops, the M2 and M4 segments, and the halothane response element to form the drug binding site of TASK-1. Our experimental data were used to validate a K(2P) open-pore homology model of TASK-1, providing structural insights for future rational design of drugs targeting K(2P) channels.