NMR analyses of the interaction between the FYVE domain of early endosome antigen 1 (EEA1) and phosphoinositide embedded in a lipid bilayer.
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.
Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
Mariko
M
Yokogawa
Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
Yoshihiro
Y
Kobashigawa
Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan.
Naoki
N
Yoshida
Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
Kenji
K
Ogura
Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan.
Kohsuke
K
Harada
Faculty of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan. Electronic address: finagaki@pharm.hokudai.ac.jp.
Fuyuhiko
F
Inagaki
D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010716:Phosphatidylinositols / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D008051:Lipid Bilayers; D010716:Phosphatidylinositols; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1