Investigation of the binding geometry of a peripheral membrane protein

Abstract

A growing number of modules including FYVE domains target key signaling proteins to membranes through specific recognition of lipid headgroups and hydrophobic insertion into bilayers. Despite the critical role of membrane insertion in the function of these modules, the structural mechanism of membrane docking and penetration remains unclear. In particular, the three-dimensional orientation of the inserted proteins with respect to the membrane surface is difficult to define quantitatively. Here, we determined the geometry of the micelle penetration of the early endosome antigen 1 (EEA1) FYVE domain by obtaining NMR-derived restraints that correlate with the distances between protein backbone amides and spin-labeled probes. The 5- and 14-doxyl-phosphatidylcholine spin-labels were incorporated into dodecylphosphocholine (DPC) micelles, and the reduction of amide signal intensities of the FYVE domain due to paramagnetic relaxation enhancement was measured. The vector of the FYVE domain insertion was estimated relative to the molecular axis by minimizing the paramagnetic restraints obtained in phosphatidylinositol 3-phosphate (PI3P)-enriched micelles containing only DPC or mixed with phosphatidylserine (PS). Additional distance restraints were obtained using a novel spin-label mimetic of PI(3)P that contains a nitroxyl radical near the threitol group of the lipid. Conformational changes indicative of elongation of the membrane insertion loop (MIL) were detected upon micelle interaction, in which the hydrophobic residues of the loop tend to move deeper into the nonpolar core of micelles. The micelle insertion mechanism of the FYVE domain defined in this study is consistent with mutagenesis data and chemical shift perturbations and demonstrates the advantage of using the spin-label NMR approach for investigating the binding geometry by peripheral membrane proteins.

FIGURE 1

Model of the FYVE domain insertion into a spherical micelle. The FYVE domain is shown in a space-filling form with the residues perturbed by the 5-doxyl spin label probe colored in gradations of red. The inositol group of the bound PI(3)P is shown as a stick model and colored blue. For any FYVE domain backbone atom i located at a distance r_i from the center of micelle O, the attenuation effect is proportional to x^-6, where x is a distance between the atom and the spin label located at a distance R from the micelle center.

FIGURE 2

The insertion interface of the FYVE domain. The three orthogonal views of the FYVE domain bound to PI(3)P- and PS- enriched DPC micelles are shown. Residues that exhibit significant signal intensity reduction upon addition of 5-doxyl spin label probe are colored red, orange and yellow for large, medium and small changes, respectively. The micelle surface is depicted as a green mesh and indicates the position of the phosphate group of DPC.

FIGURE 3

PROXYL-Pea- derivative of PI(3)P. a, Synthesis of the PROXYL-Pea- PI(3)P is shown as described in the methods. b, The histogram displays loss of amide signal intensities in the HSQC spectra caused by the addition of PROXYL-Pea- PI(3)P to the ¹⁵N-labeled FYVE domain in the presence of C₁₆-PI(3)P and d₃₈-DPC micelles. The colored bars indicate significant changes, being greater than the average plus one standard deviation. c, Residues that exhibit significant intensity reduction in (b) are labeled and colored in red, orange and yellow on the FYVE domain surface. The proxyl lipid is shown as ball-and-stick model with C, O, N and H atoms colored green, red, blue and light gray, respectively.

FIGURE 4

Comparison of the spin label experimental data with those predicted by the derived model. The histogram displays the significant amide signal intensity reductions observed in HSQC spectra (light gray) and a theoretical model (dark gray) obtained from the titration of 5-doxyl PC (5 mM) into the FYVE domain (0.2 mM) bound to C₄-PI(3)P (1 mM) and micelles formed by DPC (250 mM).

FIGURE 5

Conformational changes in the MIL of the FYVE domain upon insertion into micelles. a, The corresponding stripes derived from 3D ¹⁵N-edited NOESY spectra of 1 mM FYVE domain recorded in the absence and presence of 5 mM PI(3)P and 250 mM d₃₈-DPC. Upon insertion the intensities of a number of interresidue NOEs within the MIL are perturbed, which are highlighted by green and red boxes in (a) and indicated as green and red lines in (b) for the weakened and strengthened NOEs, respectively. b, The MIL region of the FYVE domain bound to PI(3)P (only 1-phosphate group is shown for clarity) is depicted as a gray ribbon. The MIL residues are labeled and shown as ball-and-stick models. Residues that move towards the hydrophobic core of micelles or the aqueous interface are colored orange and green, respectively.

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