© 2001 Nature Publishing Group http://structbio.nature.com news and views Solving the FYVE domain–PtdIns(3)P puzzle Paul C. Driscoll Recent crystallographic analyses of membrane-tethering FYVE finger domains from proteins involved in the regulation of endocytic vesicle trafficking have led to conflicting views of the precise nature of the contacts formed with the specific phospholipid ligand. New NMR data obtained for ligand-bound forms of a FYVE domain help resolve the atomic details of this interaction. The truly dynamic nature of eukaryotic cell membranes is brought into sharp focus by the text book description of receptor-mediated endocytosis: ∼50% of the plasma membrane is internalized and recycled every hour whereas the synthesis of new membrane is perhaps one tenth of this rate. The highly regulated process of endocytosis, by which cells recover fluid, chemicals and specific macromolecules from the external environment, is the tar- get of intense investigation. The interplay of cytosolic proteins with constituent plasma and endosomal membranes pro- vides many challenges to structural biolo- gists, not least at the interface between the soluble components and the membrane lipids themselves. Progress by the tradi- tional methods of structural investigation at this ‘phase boundary’ is particularly difficult. © http://structbio.nature.com Group 2001 Nature Publishing In a paper published recently in Science, Kutateladze and Overduin1 report an extension of their earlier work using NMR spectroscopy to analyze the lipid interactions of the FYVE protein domain from the protein early endosome Fig. 1 The chemical structure of PtdIns(3)P and the predicted ‘side-on’ interaction with the FYVE antigen-1 (EEA1). EEA1 has a major role domain of Vps27p (ref. 10; PDB code 1VFY). For the lipid, the D-myo-inositol ring is shown in red, 1′ and 3′ phosphates in blue. For Vps27p, the zinc atoms are shown in red, the ligand binding in the regulation of endosome trafficking residues of the conserved R(R/K)HHCRxCG sequence in yellow. The inner leaflet of the plasma and dynamics, specifically to promote the membrane is shown schematically in gray. tethering of two endosomal vesicles prior to fusion. The new observations serve to illuminate the likely mode of FYVE domain–membrane interfacial interac- action of class III PtdIns 3′OH-kinase. In product PtdIns(3)P is EEA1 (ref. 3). tion and provoke reassessment of alterna- yeast cells this lipid kinase is known as EEA1 contains an N-terminal zinc finger, tive predictions based upon X-ray Vps34p. Vps stands for vacuolar protein an abundance of heptad repeats predict- crystallographic studies of other mem- sorting mutant and the name reflects the ed to form a homodimeric parallel bers of this class of membrane attachment phenotype — Vps34p is essential for traf- α-helical coiled coil, a C-terminal proxi- domain. ficking of hydrolytic enzymes to the yeast mal region possibly containing a calmod- vacuole, which has a similar role to the ulin-binding IQ motif, and a C-terminal Properties of PtdIns lysosome in mammalian cells. Vps34p is Cys-rich domain with homology to sev- Phosphatidylinositol (PtdIns) provides a also implicated in the endocytotic path- eral other proteins implicated in mem- starting point for a rich chemistry that is way of yeast. Class III PtdIns 3′OH-kinase brane trafficking events. Stenmark and exploited by many different constitutive in mammalian cells is likewise now coworkers4 dubbed this last segment the and acute phase (for example, growth fac- understood to have an important role in FYVE domain based upon the four pro- tor stimulation) cellular processes2. The membrane trafficking in various con- teins then identified to contain such D-myo-inositol headgroup contains five texts3. sequences: Fab1p, YOTB, Vac1p and hydroxyl groups, three of which (posi- EEA1. The FYVE domain has eight con- tions 3′, 4′ and 5′) are known targets for FYVE domains bind PtdIns(3)P served cysteines, which coordinate two phosphorylation. In eukaryotic cells, Biochemical and inhibitor studies have Zn2+ ions, and several other conserved PtdIns(3)P (Fig. 1) is produced by the revealed that a direct target of the lipid features. The most notable of these is the nature structural biology • volume 8 number 4 • april 2001 287 © 2001 Nature Publishing Group http://structbio.nature.com news and views Fig. 2 The FYVE homodimer found in the crystal structure of the Drosophila Hrs VHS-FYVE tandem module pair12 (PDB code 1DVP), viewed from the putative ligand binding face (left). Conserved ligand binding residues are highlighted in yellow and the zinc atoms in red. The location of bound cit- rate ions (blue) is indicated by the yellow arrows. If the FYVE domains interact with PtdIns(3)P in the membrane (gray) as a homodimer, then the ori- entation of the FYVE domain with respect to the membrane (right), obtained by 90° rotation of the structure about the dotted line (left), is quite different from that predicted in ref. 9 (Fig. 1). basic amino acid sequence motif two zinc atoms and a small hydrophobic the surface of the membrane and interact R(R/K)HHCRxCG surrounding the core. The solvent-exposed face of the with the fatty acid side chains of the mem- third and fourth cysteine residues, sever- domain on the opposite side to the α-helix brane interior. al hydrophobic amino acid positions and is approximately flat and contains the The flat-face model. More recently a an isolated Arg residue towards the basic residues of the R(R/K)HHCRxCG second crystal structure of a FYVE C-terminus. motif, which are arrayed along the first domain has emerged as a part of the tan- As early as 1996 it was demonstrated strand of the first β-hairpin. dem VHS-FYVE construct of Drosophila that the endosomal localization of EEA1 A provocative feature of the intermole- Hrs13. In this structure the basic FYVE depended upon the presence of the FYVE cular contacts within these crystals is the domain architecture seen for Vps27p is domain4. Subsequently several groups occupancy of the basic surface patch pre- closely replicated, but the intriguing were able to show that fusion proteins of sented by the R(R/K)HHCRxCG motif by aspect of this study is that the protein © http://structbio.nature.com Group 2001 Nature Publishing the truncated forms of these and other the backbone and side chain carboxylate crystallizes as a homodimer, with the proteins comprising FYVE domains can groups from two residues six positions interface formed by a head-to-tail associ- directly and selectively bind PtdIns(3)P apart in the C-terminal α-helix of a lattice ation of the FYVE domain which buries in vitro and in vivo5–7. These and other neighbor. This interaction was conve- 1,920 Å2 of accessible surface area (Fig. 2). studies have led to the notion that the niently exploited to derive a molecular The dimer contact includes residues from FYVE domain, now identified in more model of how the 1,3-bisphospho-myo- the N-terminal segment and the strands than 30 different proteins, represents a inositol headgroup of PtdIns(3)P might of the two β-sheets, and includes a con- conserved adaptor domain whose prim- occupy the equivalent binding site, and served Trp residue and one of the His ary function is to act as a membrane- additionally why PtdIns(4)P or more residues in the R(R/K)HHCRxCG motif. attachment module that depends upon highly modified PtdInsPn lipids would The dimerization mode creates two interaction with PtdIns(3)P (ref. 8). not interact as a result of steric clashes. neighboring cavities, each of which con- Such a mode of PtdIns(3)P interaction tains an intimately bound citrate anion Crystal structures of the FYVE would be broadly consistent with site- from the crystallization liquor. The area domain directed mutagenesis studies which have around these ‘multianion-binding sites’ Structural biologists were not slow to clearly identified the R(R/K)HHCRxCG exhibits a very strong positive electrostat- descend on the FYVE domain as a target motif as the site of PtdIns(3)P bind- ic polarization and appears to be ideal for for structure elucidation. Misra and ing10,11. promoting an essentially flat-face interac- Hurley10 were the first to report the deter- The side-on model. In the model pro- tion of the VHS-FYVE tandem domain mination of the FYVE domain structure, posed by Misra and Hurley9 the FYVE pair with a pair of PtdIns(3)P molecules in the form of a 1.15 Å resolution crystal domain would bind the PtdIns(3)P sub- in the membrane. Moreover the apparent structure of that from yeast Vps27p strate in a ‘side-on’ manner with the long intimate association of the citrate ions (Fig. 1), which is the putative equivalent axis of the domain oriented perpendicular with the protein, combined with the simi- of the mammalian protein hepatocyte to the membrane plane (Fig. 1). These lar dimensions and spatial charge charac- growth factor-regulated tyrosine kinase authors argue that such an orientation teristics of the PtdIns(3)P headgroup substrate (Hrs) and plays a role in endo- would place the loop containing the con- again conveniently allowed for modeling some maturation9. The 60-residue frag- served hydrophobic amino acids (includ- of the putative physiological interaction. ment is folded into a pair of anti-parallel ing a di-leucine pair in the case of However the outcome of this exercise is a β-hairpins and a C-terminal α-helix that, Vps27p), which occurs just upstream of position for the 1,3-bisphospho-myo- together with an N-terminal region of the β1 strand in the FYVE domain con- inositol headgroup that is rather different irregular secondary structure, encloses the sensus, in an ideal position to penetrate from that anticipated from the Vps27p 288 nature structural biology • volume 8 number 4 • april 2001 © 2001 Nature Publishing Group http://structbio.nature.com news and views Fig.