Abstract
Phosphoinositides (PIs) are crucial lipid components of membranes and are involved in a number of cellular processes through interactions with their effector proteins. Recently, we have established a lipid-protein nanoscale bilayer (nanodisc) containing PIs, hereafter referred to as PI-nanodisc and demonstrated that it could be used for both qualitative and quantitative evaluations of protein-membrane interactions. Here, we report further NMR analyses for obtaining structural insights at the residue-specific level between PI-binding effector protein and PI-nanodisc, using the FYVE domain of early endosome antigen 1 (EEA1), denoted as EEA1 FYVE, and PI(3)P-nanodisc as a model system. We performed a combination of the NMR analyses including chemical shift perturbation, transferred cross-saturation, and paramagnetic relaxation enhancement experiments. These enabled an identification of the interaction surface, structural change, and relative orientation of EEA1 FYVE to the PI(3)P-incorporated lipid bilayer, substantiating that NMR analyses of protein-membrane interactions using nanodisc makes it possible to show the residue-specific interactions in the lipid bilayer environment.
Highlights
Interaction between phosphoinositides (PIs) and effector proteins has not been fully elucidated in a lipid bilayer environment
These enabled an identification of the interaction surface, structural change, and relative orientation of endosome antigen 1 (EEA1) FYVE to the PI[3]P-incorporated lipid bilayer, substantiating that NMR analyses of protein-membrane interactions using nanodisc makes it possible to show the residue-specific interactions in the lipid bilayer environment
PI[3]Pnanodisc was applied to characterize the physiological features of the interaction between EEA1 FYVE and PI[3]P embedded in a lipid bilayer environment, which led to a model consistent with the relative orientation suggested by the crystal structure of the dimeric C-terminal domain of EEA1 containing the coiled-coil and FYVE domains [17]
Summary
Interaction between phosphoinositides (PIs) and effector proteins has not been fully elucidated in a lipid bilayer environment. We performed a combination of the NMR analyses including chemical shift perturbation, transferred cross-saturation, and paramagnetic relaxation enhancement experiments These enabled an identification of the interaction surface, structural change, and relative orientation of EEA1 FYVE to the PI[3]P-incorporated lipid bilayer, substantiating that NMR analyses of protein-membrane interactions using nanodisc makes it possible to show the residue-specific interactions in the lipid bilayer environment. PI[3]Pnanodisc was applied to characterize the physiological features of the interaction between EEA1 FYVE and PI[3]P embedded in a lipid bilayer environment, which led to a model consistent with the relative orientation suggested by the crystal structure of the dimeric C-terminal domain of EEA1 containing the coiled-coil and FYVE domains [17]. The evaluation of the membrane-protein interactions shown by nanodisc-based NMR analyses is a feasible approach for validation of physiological PI-effector protein interactions
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