Phosphatidylinositol 3-Phosphate Recognition by the FYVE Domain Tatiana G Kutateladze, Kenyon D Ogburn, William T Watson, Tonny de Beer, Scott D Emr, Christopher G Burd, Michael Overduin  Molecular Cell  Volume 3, Issue 6, Pages 805-811 (June 1999) DOI: 10.1016/S1097-2765(01)80013-7

Figure 1 Identification of the EEA1 FYVE Domain Residues that Bind Specifically to PtdIns(3)P (A) Twenty amino acids display large chemical shift changes upon PtdIns(3)P binding. Six 1H-15N HSQC spectra of uniformly 15N-labeled FYVE domain (0.25 mM) are superimposed and color coded as shown in the inset according to the concentration of unlabeled dibutanoyl PtdIns(3)P (shown in magenta with R = C4H9). Residues exhibiting changes larger than those indicated by the dotted lines in Figure 1B are labeled, as are those involved in interactions with micelles. The boxed cross peaks are indicated at a lower contour level. The majority of the resonances of the zinc-bound FYVE domain were assigned: 90% of the protons attached to carbon atoms, 84% of the detectable 13C resonances, and 97% of the backbone amide 15N resonances. (B) The PtdIns(3)P, PtdIns(5)P, and PtdIns–induced perturbations of 15N and 1H resonances are compared. Absolute changes in each residue’s backbone amide chemical shifts caused by addition of the indicated amount of dibutanoyl PtdIns(3)P (magenta), PtdIns(5)P (green), and PtdIns (orange) are shown. Residues for which the changes due to PtdIns(3)P or PtdIns(5)P addition could not be measured due to lack of sensitivity and/or line broadening are indicated by † and §, respectively. Due to their extreme pH sensitivity, chemical shifts of residues marked by ‡ were corrected. Molecular Cell 1999 3, 805-811DOI: (10.1016/S1097-2765(01)80013-7)

Figure 2 Secondary Structure and Alignment of the FYVE Domain Sequences (A) Schematic drawing of the four β strands (β1, β2, β3, and β4) that form two β hairpins depicted as yellow arrows. The α helix is depicted as a yellow cylinder, and loops are shown as blue lines. Residues that exhibit substantial chemical shift changes due to PtdIns(3)P binding, as shown in Figure 1B, are labeled in magenta. Residues labeled in green are influenced by membrane interactions. Cysteines that are predicted to coordinate the two zinc ions are labeled in blue. NMR structural information used to define the structure include interproton distances from nuclear Overhauser effects (dashed orange lines), dihedral bond angles, and chemical shift indices. (B) Binding site residues and secondary structure elements in 18 FYVE domains are conserved. Residues involved in PtdIns(3)P, membrane, and zinc binding are shown in magenta, green, and blue type, respectively, and other residues at conserved hydrophobic positions are in orange. Every tenth residue in the EEA1 FYVE domain is capped with a black dot, and seven EEA1 residues that have been mutated are indicated by red boxes. Deletions added for clarity are indicated by brackets. The PtdIns(3)P-induced NMR shifts and secondary structure are shown above and below the sequence, respectively. The EEA1, Hrs, SARA, FDP, BK085E05, and KIAA0647 protein sequences are human forms; Ankhzn and p235 are from mouse; YOTB, T10G3.5, F01F1.4, F22G12, T23B5, and MHP1 are from Caenorhabditis elegans; and Vac1p, Fab1p, and Vps27p are from Saccharomyces cerevisiae. Molecular Cell 1999 3, 805-811DOI: (10.1016/S1097-2765(01)80013-7)

Figure 3 In Vivo Localization of Mutant EEA1(FYVE) Domains Expression vectors encoding GFP fused to wild-type EEA1 FYVE domain (amino acids 1305–1410), or two mutant FYVE domains (mutations indicated), were introduced into wild-type yeast cells. Living cells were examined by fluorescence microscopy (left) and by DIC optics (right). The wild-type GFP-EEA1(FYVE) protein localizes predominantly to endosomes, while each of the mutant proteins are localized to the cytosol, indicating that they are defective in PtdIns(3)P binding. Molecular Cell 1999 3, 805-811DOI: (10.1016/S1097-2765(01)80013-7)

Figure 4 The EEA1 FYVE Domain Unfolds Reversibly When Zinc Is Removed Shown are four NMR spectra of the same FYVE domain sample that were collected after EDTA was added stepwise to remove bound zinc, and after zinc was subsequently added back to the sample. (A) The dispersed scatter of 1H-15N cross peaks in the HSQC spectrum reflects the structured state of the FYVE domain (0.25 mM). Peaks of the backbone 1H-15N amides of eight conserved cysteines predicted to coordinate zinc are colored blue, and those derived from the 20 N-terminal residues are green. A diagram of the zinc coordination sites is shown. (B) After addition of equimolar EDTA (0.25 mM) to the FYVE domain, the 1H-15N cross peaks representing the structured state decrease in intensity, and a second set of cross peaks concentrated near the center of the spectrum appears. The intensity of the peaks of the eight cysteines in the structured state (blue) diminishes to the same extent, indicating that the eight cysteines release the two zinc ions simultaneously. The Trp-1348 side chain exhibits two 1H-15N peaks corresponding to the structured and unstructured state, respectively. (C) After addition of excess EDTA (0.75 mM), the 1H-15N cross peaks corresponding to the structured state disappear entirely while peaks corresponding to the unstructured state increase in intensity. Those peaks derived from the 20 N-terminal residues (green) do not move substantially, indicating that these residues are not involved in zinc-dependent structure. (D) The structure is restored by addition of ZnSO4 (0.75 mM) to the FYVE domain (0.25 mM). Molecular Cell 1999 3, 805-811DOI: (10.1016/S1097-2765(01)80013-7)