Solving the FYVE Domain–Ptdins(3)P Puzzle

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 target of intense investigation. The interplay of cytosolic proteins with constituent plasma and endosomal membranes provides many challenges to structural biologists, 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

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 citrate 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 orientation 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

Fig. 3 The solution structure of the EEA1 FYVE domain obtained by Kutateladze and Overduin1 coordinates kindly provided by the authors prior to publication) with the headgroup of PtdIns(3)P (magenta) bound to the conserved ligand binding residues (yellow), as obtained from NMR data 1364 with soluble di-C4-PtdIns(3)P. Residues from the Phe -Ser-Val-Thr-Val loop that have NMR resonances that are strongly perturbed by interaction with mixed micelles (gray) are shown in white.

structure. While the positions of the micelles, NMR resonances corresponding 3-phosphates in each model are broadly to the N-terminal loop Phe1364-Ser-Val- superimposed, the locations of the Thr-Val are strongly broadened and shift- 1-phosphate in each model are far apart: ed. Such spectral changes are much the dimer contacts in the VHS-FYVE pro- stronger when PtdIns(3)P is included in vide a steric barrier to positioning the the micelles. This region of EEA1 corre- phosphoinositol ring in the binding sponds to the di-leucine-containing loop mode suggested by modeling with the predicted to be membrane-proximal for monomeric Vps27p domain. This model the Vps27p FYVE domain, and the suggests that PtdIns(3)P signaling might authors conclude that the hydrophobic require dimerization of the target FYVE- side chains of this region insert into the domain containing protein. micelle surface (Fig. 3), in a similar mode sity of NMR parameters. The complex to that predicted by Misra and Hurley9. structure does not have the high resolu- Application of NMR spectroscopy It is notable that these experiments are tion necessary to dissect the precise How to resolve the differences between the interpretable at all since NMR investiga- nature of the intermolecular interaction respective modeling exercises? Cry- tions of micelle-bound proteins are noto- but the outcome is sufficient to reveal a stallization of peripheral membrane pro- riously difficult, and do not always yield binding orientation that is very similar to teins in contact with membrane bilayers complexes that provide high resolution that predicted for Vps27p (the authors remains a fantastical prospect. Co-crystal- spectra, although there are notable excep- perhaps overstate the differences), and lization of any FYVE domain construct tions13,14. That such studies have worked at that is therefore quite dissimilar to the with a soluble PtdIns(3)P mimic has not all may point to a very dynamic nature of model predicted for Hrs FYVE (and for yet proved possible. Happily the path the protein lipid interface, such that the which it is less obvious how to account for taken by Overduin and colleagues with effective correlation time of the micelle- the observations that the hydrophobic their heteronuclear NMR investigation of bound protein is not as long as might be residues in the pre-β1 loop are shifted by the FYVE domain of EEA1 provides anticipated for a rigid complex. the presence of the lipid micelles). experimental illumination of this crystal- Interestingly, the interaction between © http://structbio.nature.com Group 2001 Nature Publishing lographic impasse. membrane-bound PtdIns(3)P and EEA1 A ligand-dependent conformational 1 Their recent work describes the full FYVE is predicted to be quite strong (Kd change? solution structure determination of both ∼50 nM). However the NMR titration While on its own chemical shift mapping the free and the di-C4-PtdIns(3)P-bound experiments have the characteristic of of protein–ligand interactions is a low EEA1 FYVE domain (Fig. 3). The report intermediate-fast exchange on the chemi- resolution method of assessing the nature also builds upon previous experiments in cal shift time-scale, which implies a rather of protein–ligand interfaces, even when which this group examined the effects of short residence time for the protein at a the preliminary reports of the FYVE- titrating 15N-labeled EEA1 FYVE domain given site on the micelle. lipid titration were reported, Overduin with PtdIns, PtdIns(3)P and PtdIns(5)P The dynamic nature of the FYVE and colleagues suspected that the wide- both in short chain (di-butanoyl) soluble domain–micelle interface severely com- spread distribution of PtdIns(3)P- form or long chain (di-palmitoyl) form plicates the process of obtaining the induced spectral perturbations pointed while embedded in diphosphatidyl- structure of the micelle-bound form of towards a ligand-dependent conforma- choline (DPC) micelles10. The most pro- the protein. As a compromise, tional change in the protein. The com- nounced effects in each case were Kutateladze and Overduin1 determined parative analysis of free and bound forms observed with the 3-phosphorylated the solution structures of both lipid-free of the EEA1 FYVE domain now reveals lipids. With di-C4-PtdIns(3)P, fast and di-C4-PtdIns(3)P-bound forms of elevated ordering of the N-terminal seg- exchange chemical shift perturbations EEA1 FYVE based upon standard NOE ment and the slight repositioning of cer- were observed for amide NH signals of 20 and torsion angle restraint data, coupled tain structural elements that are residues. The largest effects map to sur- with — for the complex — an interpreta- described as modifying the lipid head- face patch on the FYVE domain that com- tion of likely hydrogen bond interactions group binding pocket and enhancing the prises the N-terminal loop segment, the between the phosphate groups of the lig- packing and stability of the FYVE β1-loop-β2 segment encompassing the and and those groups that displayed the domain in the bound state. Certainly R(R/K)HHCRxCG motif and the imme- most pronounced chemical shift pertur- there is a greater number of NOE effects diate vicinity of the conserved Arg in bations in the ligand titrations. Such a that could be exploited for structure cal- β-strand 4. These results nicely corrobo- strategy is enforced partly by the small culations, which points to such changes rate the biochemical data that predicted number of NOE contacts between the taking place. that the same face of the molecule is the protein and lipid, and also because the By the nature of NMR-based solution site of PtdIns(3)P binding. fundamentally important interactions structure determinations it is difficult to Intriguingly Kutateladze and Overduin1 operate via the proton-poor phosphate control precisely for the presence of the comment that in the experiments with groups, which do not yield up a high den- lipid ligand and changes in the rotational

correlation time in such comparisons, domain, and on the presence on the endo- University College London, Gower Street, and arguably one runs the risk of over- some of a functional Rab5 protein, a small London WC1E 6BT, UK, and the Ludwig stating the conformational differences of GTPase of the Ras superfamily that in the Institute for Cancer Research, 91 Riding what are — by the standards of crystallo- GTP-bound state can bind to this region House Street, London W1W 7BS, UK. graphers at least — medium resolution of the EEA1 protein15,16. The relatively low email: [email protected] structures. Nevertheless the authors of affinities of the EEA1 Rab5-binding 1. Kutateladze, T.G. & Overduin, M. Science 291, this study appear confident that these domain for Rab5–GTP and of the FYVE 1793–1796 (2001). results point to a structural plasticity of domain itself for PtdIns(3)P suggest that, 2. Leevers, S.J., Vanhaesebroeck, B. & Waterfield, M.D. Curr. Opin. Cell Biol. 11, 219–225 (1999). the FYVE domain that may be relevant under physiological conditions, the EEA1 3. Corvera, S., D’Arrigo, A. & Stenmark, H. Curr. Opin. for the regulation of EEA1 function. protein is recruited only to membranes Cell Biol. 11, 460–465 (1999). 4. Stenmark, H., Aasland, R., Toh, B.H. & D’Arrigo, A. Alternative approaches that might that contain both of these components. A J. Biol. Chem. 271, 24048–24054 (1996). address this issue include the use of more complete understanding of the 5. Gaullier, J.M. et al. Nature 394, 432–433 (1998). 6. Patki, V., Lawe, D.C., Corvera, S., Virbasius, J.V. & calorimetric and nuclear relaxation mea- structural basis of the EEA1 FYVE domain Chawla, A. Nature 394, 433–434 (1998). surements, as well as crystallization of interaction with membrane bilayers may 7. Burd, C.G. & Emr, S.D. Mol. Cell 2, 157–162 (1998). 8. Stenmark, H. & Aasland, R. J. Cell Sci. 112, the lipid bound state (though this runs therefore require the construction of a 4175–4183 (1999). the risk of appearing to freeze out ternary complex of Rab5–GTP–EEA1 9. Misra, S. & Hurley, J.H. Cell 97, 657–666 (1999). 10. Kutateladze, T.G. et al. Mol. Cell 3, 805–811 (1999). dynamic disorder). C-terminus–PtdIns(3)P. Such a goal pre- 11. Gaullier, J.M., Ronning, E., Gillooly, D.J. & Stenmark, sents a more challenging target to struc- H. J. Biol. Chem. 275, 24595–24600 (2000). 12. Mao, Y. et al. Cell 100, 447–456 (2000). The bigger picture tural biologists, particularly if we would 13. Van den Berg. B. et al. Nature Struct. Biol. 2, In this vein it is worthwhile to note that in like to better understand the role of the 402–406 (1995). 14. Xu, R.X., Pawelczyk, T., Xia, T.H. & Brown, S.C. vivo labeling of early endosomes with membrane itself. Biochemistry 36, 10709–10717 (1997). tagged-FYVE proteins depends upon the 15. Simonsen, A. et al. Nature 394, 494–498 (1998). 16. Lawe, D.C., Patki, V., Heller-Harrison, R., Lambright, inclusion of the amino acid sequence Paul C. Driscoll is in the Department of D. & Corvera, S. J. Biol. Chem. 275, 3699–3705 immediately upstream of the FYVE Biochemistry and Molecular Biology, (2000).

Making the most of metal ions

Crystal structures of the homing endonuclease I-CreI bound to substrate DNA and divalent metals show that one metal ion is shared between the two active sites of the enzyme. This arrangement appears uniquely suited to the formation of double-stranded DNA breaks via a concerted reaction.

Many group I introns possess open read- order to avoid generating multiple dou- Metal ions in biological reactions ing frames which encode DNA endo- ble-stranded breaks within the host The importance of divalent metal cations nucleases that promote the mobility of genome. On the basis of conserved in promoting biologically important the intron in a process known as hom- sequence motifs, group I homing phosphoryl transfer reactions has been ing1. The introns exhibit widespread enzymes fall into four structural families. appreciated for some time. Recognition phylogenetic diversity, and have now The largest of these, the LAGLIDADG of the central role of ATP hydrolysis in been found in all three biological family, is the best studied and is now rep- biochemistry led to early emphasis on domains. In the homing mechanism, a resented by crystal structures of the understanding the mechanisms for donor allele possessing the intron (I+) is I-CreI, PI-SceI, I-DmoI and PI-PfuI accelerating hydrolysis of phospho- paired with a recipient cognate allele that enzymes2–5. However, the catalytic mech- monoesters7–9. Model reactions catalyzed lacks the element (I–). The intron-encod- anisms of these homing endonucleases by small organic compounds, together ed homing endonuclease cleaves both have remained obscure. A significant step with enzymatic studies and theoretical strands of the I– allele at the splice site, in understanding the basis for rate approaches, have shown that metal ions initiating a duplication of the intron fol- enhancement is now reported on page can play important roles in NTP cleavage lowed by resolution via a double-strand- 312 of this issue of Nature Structural reactions10-15. These reactions are neces- ed break repair pathway (Fig. 1). Thus, Biology. Based on the high-resolution sary for energy conversion, regulation of the homing endonuclease gene functions structures of two ternary enzyme–DNA– protein activity and cell signaling process- in a selfish manner, ensuring propagation metal ion complexes, Chevalier et al.6 es. By contrast, enzymes that hydrolyze by virtue of the ability of its protein propose a novel DNA cleavage mecha- phosphodiesters instead are central to product to initiate recombination events. nism for I-CreI in which two catalytic replication, transcription, recombina- The homing endonucleases must be active sites share a total of three metal tion, and DNA repair through their highly specific for their target sites in ions. manipulation of DNA and RNA.

290 nature structural biology • volume 8 number 4 • april 2001