Phosphatidylinositol 3-Phosphate Recognition by the FYVE Domain

Molecular Cell, Vol. 3, 805–811, June, 1999, Copyright 1999 by Cell Press

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,† and Michael Overduin*§ * Department of Pharmacology University of Colorado Health Sciences Center Denver, Colorado 80262 † Department of Cell and Developmental Biology and Institute for Human Gene Therapy University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104 ‡ Division of Cellular and Molecular Medicine Howard Hughes Medical Institute University of California, San Diego School of Medicine La Jolla, California 92093-0668

Summary Recognition of phosphatidylinositol 3-phosphate (PtdIns(3)P) is crucial for a broad range of cellular signaling and membrane trafficking events regulated by phosphoinositide (PI) 3-kinases. PtdIns(3)P binding by the FYVE domain of human early endosome autoantigen 1 (EEA1), a protein implicated in endosome fusion, involves two b hairpins and an a helix. Specific amino acids, including those of the FYVE domain’s conserved RRHHCRQCGNIF motif, contact soluble and micelleembedded lipid and provide specificity for PtdIns(3)P over PtdIns(5)P and PtdIns, as shown by heteronuclear magnetic resonance spectroscopy. Although the FYVE domain relies on a zinc-binding motif reminiscent of RING fingers, it is distinguished by novel structural features and its PtdIns(3)P-binding site.

Introduction PI kinases regulate cellular growth and differentiation, apoptosis, and cytoskeletal rearrangement. These enzymes produce phosphorylated PtdIns’s, which act as second messengers that draw protein complexes to subcellular compartments and influence their activities (Wurmser et al., 1999). The distribution of PtdIns(3)P is restricted by the location of phosphoinositide (PI) 3-kinases, allowing interacting proteins to be selectively recruited to specific membrane compartments. Cytoplasmic proteins specifically recognize phosphatidylinositol 3-phosphate (PtdIns(3)P) via a conserved signaling module called the FYVE domain (Burd and Emr, 1998; Gaullier et al., 1998; Patki et al., 1998). PtdIns(3)P recognition appears to be the exclusive responsibility of FYVE domains since C2, pleckstrin homology (PH), and src homology 2 (SH2) domains bind other phospholipids and multiply phosphorylated PtdIns’s but not PtdIns(3)P (reviewed in Bottomley et al., 1998). § To whom correspondence should be addressed (e-mail: michael.

[email protected]).

The in vivo interactions between FYVE domains and PtdIns(3)P are essential for the function of several proteins. For example, EEA1 (human early endosome autoantigen 1) is recruited to endosomes by PtdIns(3)P, where it forms an important component of the membrane fusion machinery (Mills et al., 1998; Simonsen et al., 1998; Christoforidis et al., 1999). EEA1 and its yeast ortholog, Vac1p, bind the activated form of the Rab5 GTPase, and Vac1p binds a t-SNARE vesicle receptor and the Sec1p family protein Vps45p (Simonsen et al., 1998; Peterson et al., 1999). Thus, these FYVE domain proteins directly regulate the core membrane fusion machinery. Other yeast FYVE domain proteins that are involved in vesicular traffic are Vps27p, which sorts endocytic and biosynthetic cargo (Piper et al., 1995), and Fab1p, a PtdIns(3)P 5-kinase that regulates vacuole size and cargo-selective sorting (Odorizzi et al., 1998). Organelle-specific targeting is provided by the FYVE domain of SARA, an adaptor protein that recruits the transcription factors to the activated transforming growth factor b receptor (Tsukazaki et al., 1998). The FYVE and PH domains of FGD1, an exchange factor in Rac/Rho pathways that is defective in the human disease faciogenital dysplasia, link PI kinase signaling to actin cytoskeletal dynamics (Pasteris et al., 1994). Thus, FYVE domains are PtdIns(3)P-binding modules found in essential components of a wide variety of cellular pathways. The exclusive specificity of the FYVE domain for PtdIns(3)P is reflected by its novel sequence. Unlike other domains that bind PtdIns derivatives, the FYVE domain requires two zinc ions coordinated by eight cysteines (Stenmark et al., 1996). FYVE domain binding to PtdIns(3)P is abolished by cysteine mutations or zinc chelation (Burd and Emr, 1998; Gaullier et al., 1998). FYVE domain family members share z40% sequence identity (Shisheva et al., 1999) and are defined by an RRHHCRXCG motif (where X is any residue) required for PtdIns(3)P interaction (Burd and Emr, 1998) and a conserved region N-terminal to the first cysteine (Stenmark et al., 1996). The functional and structural importance of these residues is characterized using the FYVE domain of EEA1, the prototypic member of this family of signaling domains. Results and Discussion The PtdIns(3)P-Binding Site PtdIns(3)P is bound by the FYVE domain through an extensive basic interface, as identified by NMR (Figure 1). The most important binding element is the RRH HCRQCGNIF sequence (EEA1 residues 1369–1380) whose 1 H/15N resonances exhibit the largest changes upon interaction of the FYVE domain with a water-soluble dibutanoyl form of PtdIns(3)P. Six basic residues, Lys-1347, Arg-1369, Arg-1370, His-1372, Arg-1374, and Arg-1399, are particularly sensitive to PtdIns(3)P binding. Consequently, their basic side chains are strong candidates for directly binding phosphate groups of the lipid. The apolar nature of residues Met-1358, Ile-1379, and Phe1380 suggests that they form hydrophobic interactions

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large number of chemical shift changes found in the atoms of amino acids throughout the structure. The EEA1 FYVE domain contains four b strands and an a helix, all of which contribute to PtdIns(3)P binding by direct contacts or indirect structural changes, as shown by NMR chemical shift perturbations (Figures 1B and 2A). In addition, three N-terminal residues, Asp1351, Val-1354, and Asn-1356, pack against Ile-1379 based on long-range nuclear Overhauser effects (NOEs). This structural element may orient the nearby Lys-1347, Asn-1352, Glu-1353, and Gln-1355 residues for PtdIns (3)P interaction. The loop preceding b1 contains the PheSerValThr-1367 residues, which we predict to interact with the membrane. The first two strands, b1 and b2, form a hairpin that is centrally involved in PtdIns(3)P binding. The second b hairpin is irregular, containing a conserved proline residue and backbone angles at Arg1399 that are not typical of a b strand. Interestingly, the latter residue appears to bind directly to PtdIns(3)P. The C-terminal a helix is amphipathic with Phe-1405 and Leu-1408 packing against the hydrophobic core.

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 5 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 15 N resonances. (B) The PtdIns(3)P, PtdIns(5)P, and PtdIns–induced perturbations of 15 N 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.

with the lipid, inducing substantial 1H/15N chemical shift perturbations. Conformational changes are likely to occur when the FYVE domain binds PtdIns(3)P due to the

Identification of a Membrane Insertion Element The interactions of the FYVE domain with PtdIns(3)Pembedded lipid micelles were investigated by NMR to identify the elements that insert into cellular membranes. The PheSerValThr-1367 sequence is unusual in that its 1 H/15N resonances disappear upon addition of dipalmitoyl PtdIns(3)P embedded in dodecylphosphocholine (DPC) micelles (data not shown). This is in contrast to the absence of similar changes in the NMR signals of these residues when soluble PtdIns(3)P is added (Figure 1A). In addition, Gly-1377’s resonances move significantly only upon titration with micelle-embedded PtdIns(3)P. Thus, these residues are predicted to be involved in membrane interactions based on their micelle-specific NMR spectral changes. In fact, it is likely that the hydrophobic side chains of Phe-1364, Val-1366, and Thr-1367 insert directly into the lipid bilayer. Otherwise, the FYVE domain binds PtdIns(3)P-containing micelles in a manner that mirrors the interactions with dibutanoyl PtdIns(3)P since similar 1H/15N chemical shift changes are observed. These results emphasize that the FYVE domain not only specifically binds the inositol ring phosphorylated at the 3 position, but also interacts directly with membranes. Mutating the PtdIns(3)P and Membrane-Binding Residues Disrupts Localization The activities of mutant FYVE domains in which residues implicated in lipid binding are substituted were tested using in vitro liposome binding assays and in vivo assays of PI 3-kinase-dependent localization. A FYVE domain with a mutated RRHHCR sequence does not bind PtdIns(3)P (Burd and Emr, 1998). Moreover, mutation of either of two conserved residues predicted to directly contact PtdIns(3)P, Arg-1374 and Arg-1399, to a glycine abolishes PtdIns(3)P-dependent liposome binding and localization (Figure 3). The NMR data also suggest that many other residues contribute to PtdIns(3)P binding, and this conclusion is corroborated by two other mutants. A random mutagenesis screen was performed to obtain EEA1 mutants with subtle defects in cellular

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Figure 2. Secondary Structure and Alignment of the FYVE Domain Sequences (A) Schematic drawing of the four b strands (b1, b2, b3, and b4) that form two b hairpins depicted as yellow arrows. The a 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.

localization in vivo. Two FYVE domain mutants were found, Asp-1351→Val and Asn-1356→Asp, which partially localize to the cytosol when expressed as fusions to green fluorescent protein (data not shown). In light of the NMR structural data, these conserved, polar amino acids stabilize neighboring PtdIns(3)P-binding residues. The NMR experiments suggest an unanticipated role in membrane insertion by the PheSerValThr-1367 sequence. The ValThr-1367 residues were mutated to GlyGly and GluGlu, respectively, and both mutants were found to be completely defective in PtdIns(3)P-dependent localization in vivo (Figure 3) and in vitro (data not shown), indicating that these residues provide an essential interaction with PtdIns(3)P-containing membranes. Taken together, the mutagenesis results support the conclusion that an extensive binding site involving the majority of the FYVE domain structure mediates specific recognition of PtdIns(3)P-containing membranes. Specificity for PtdIns(3)P The ability of the FYVE domain to specifically recognize PtdIns(3)P was characterized by comparing the different phospholipid interactions by NMR. PtdIns(3)P is bound

with much higher affinity than either PtdIns(5)P or PtdIns. The PtdIns(3)P interaction involves a mM KD and a relatively slow off-rate since the bound and free states exhibit intermediate to fast exchange on the NMR time scale (Figure 1A). In particular, NMR signals from several residues including Gln-1355, Met-1358, His-1371, Cys1373, and Phe-1380 exhibit considerable line broadening while those of Asn-1352 and His-1372 also display multiple NMR peaks upon PtdIns(3)P titration. The interaction with PtdIns(5)P was also tested since this naturally occurring lipid is structurally most similar to PtdIns(3)P. The PtdIns(5)P interaction is much weaker and is characterized by fast exchange kinetics (data not shown) and less extensive chemical shift changes. The unphosphorylated inositol ring does not exhibit significant binding since addition of even a 3-fold excess of PtdIns fails to induce substantial chemical shift changes in the FYVE domain (Figure 1B). Thus, PtdIns(3)P is the preferred ligand of FYVE domains. The same set of residues that bind PtdIns(3)P serves to bind PtdIns(5)P (Figure 1B). Both lipids influence the 1 H/15N resonances of His-1372, Arg-1374, and Arg-1399, indicating that these residues interact with either PtdIns

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(Ds) of the protein measured by NMR was used to estimate the oligomeric state (Altieri et al., 1995). The average apparent molecular weight of the 9.7 kDa FYVE domain at concentrations of 0.2 and 2 mM was estimated to be approximately 15 and 19 kDa, respectively. These results are consistent with an equilibrium in which dimers are present at protein concentrations of at least 0.2 mM. Indeed, only monomers are detected at low protein concentration (10 mM) by sedimentation velocity experiments. The FYVE domain dimers are predicted to be in fast exchange with the monomeric state since only a single set of cross peaks is detected by NMR at protein concentrations from 150 mM to 2 mM. Dimers of the FYVE domain bind more tightly to PtdIns(3)P-bearing membranes than monomers. When FYVE domain dimerization is induced by fusion to glutathione S-transferase, we observe significantly greater binding to PtdIns(3)P liposomes than with FYVE domains in untagged or His6-tagged forms (data not shown). The region immediately N-terminal to the FYVE domain may contribute to dimerization since a longer EEA1 construct that includes residues 1305–1324 has higher affinity for liposomes containing PtdIns(3)P. Thus, FYVE domain dimerization may reinforce the adjacent dimer interface formed by EEA1’s central a-helical heptad repeat region. In addition, dimerization could influence interactions with Rab5, which binds EEA1 immediately N-terminal to its FYVE domain (Simonsen et al., 1998).

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.

derivative. The backbone 1H-15N resonances of His-1371 disappear entirely upon PtdIns(3)P titration and exhibit large chemical shift changes upon PtdIns(5)P addition, supporting its critical involvement in binding either lipid. However, the chemical shifts of three basic residues (Lys-1347, Arg-1369, and Arg-1370) are much less influenced by PtdIns(5)P than by PtdIns(3)P. Consequently, these residues are likely to directly contact the phosphate group at the 3 but not the 5 position of the inositol ring, determining the specificity of the FYVE domain for PtdIns(3)P.

The FYVE Domain Forms Functional Dimers Cytosolic EEA1 has been shown to form parallel coiledcoil homodimers that would juxtapose the C-terminal FYVE domains (Callaghan et al., 1999). Weak dimerization of EEA1’s FYVE domain is evident at high protein concentrations. The translational diffusion coefficient

Zinc Is Required for FYVE Domain Structure Although FYVE domains are known to require a pair of zinc ions for PtdIns(3)P binding and membrane interactions (Stenmark et al., 1996; Burd and Emr, 1998; Gaullier et al., 1998), the structural effects of zinc are unknown. In order to define the role of zinc, NMR spectra were collected after addition of ethylenediaminetetraacetic acid (EDTA) into a FYVE domain sample. As zinc is progressively removed by the chelator, resolved 1H-15N cross peaks derived from the domain’s structured state diminish in intensity, including those of all eight cysteines predicted to coordinate zinc (Figures 4A and 4B). At the same time, a second set of cross peaks corresponding to an unstructured state appears with increasing intensity (Figures 4B and 4C). The conversion of the former resolved peaks into the latter upon addition of excess chelator indicates that essentially all the protein molecules have unfolded. Similar effects are seen with the chelator N,N,N9,N9-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), confirming that the FYVE domain is structured only in the presence of zinc. The loss of structure is reversible (although not completely), since the reintroduction of zinc restores the structure as evidenced by the reappearance of the dispersed cross peaks stemming from the folded state (Figure 4D). The zinc-dependent structure of the FYVE domain begins at Arg-1346. The resonances of the preceding, N-terminal amino acids in the FYVE domain construct are not perturbed by zinc removal (Figure 4). These residues appear to be unstructured due to their poorly resolved chemical shifts (Figure 4C) and lack of NOEs to the FYVE domain. The rest of the protein is reversibly unfolded upon zinc removal and binds zinc tightly based on the slow exchange of the zinc-bound and free states (Figure 4).

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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 zincdependent structure. (D) The structure is restored by addition of ZnSO4 (0.75 mM) to the FYVE domain (0.25 mM).

The structure of the FYVE domain is similar to rabphilin3A’s zinc-binding domain (Ostermeier and Bru¨nger, 1999) but bears little resemblance to C2, PH, and SH2

domains beyond a clustering of basic residues that bind phospholipids (Bottomley et al., 1998). The positions of the strands and helix are conserved between the EEA1 FYVE domain and rabphilin-3A. Zinc is braced within a tetrahedral coordination geometry by rabphilin-3A’s two CX2CX13,14CX2C motifs that closely match EEA1’s sequence. A homologous coordination of one zinc ion would involve EEA1 cysteines 1357, 1360, 1381, and 1384, thus stabilizing the b1/b2 hairpin. A second zinc site is predicted to be formed by cysteines 1373, 1376, 1401, and 1404. However, FYVE domains are distinguished from rabphilin-3A, which has not been shown to bind to lipids or membranes, by the presence of the RRHHCRXCG motif. More distantly related are three RING finger structures (Barlow et al., 1994; Borden et al., 1995; Bellon et al., 1997), which also share the FYVE domain’s zinc coordination pattern but differ structurally. Some RING fingers and rabphilin-3A’s zinc-binding domain mediate protein–protein interactions or act as structural supports for other, functional domains. In contrast, FYVE domains are unique members of an extensive family of zinc-binding domains that have a clearly conserved biological function, PtdIns(3)P recognition. The crystal structure of the lipid-free Vps27p FYVE domain has recently been completed (Misra and Hurley, 1999), and despite its overall similarity to the EEA1 FYVE domain, several important differences exist. Our NMR study of EEA1 includes the entire FYVE domain and 22 additional N-terminal amino acids. Residues in this region such as Arg-1346 exhibit significant chemical shift changes upon PtdIns(3)P addition (Figure 1B) and are likely to be involved in lipid recognition. The two structures differ in the region immediately preceding the first zinc coordination site. A nonstandard N-terminal b strand seen in the Vps27p structure is absent in EEA1. The NMR data shows instead that the FYVE domain’s N terminus packs against the hydrophobic core and excludes main chain hydrogen bonds between residues corresponding to Ile-171 and Gly-196 of Vps27p. In addition, the positions of several b strands (especially b2) differ from those predicted for EEA1 from the Vps27p structure. The last two residues of EEA1 (GlnGly-1410) are part of the FYVE domain structure, exhibiting NOEs to the rest of the domain. The corresponding residues are absent from the Vps27p construct, which instead includes a six-residue vector-encoded C-terminal extension that forms an unphysiological contact. Finally, Vps27p’s sequence is unusual among FYVE domains while a classical zinc coordination site involving eight cysteines is found in EEA1. The EEA1 FYVE domain binds both PtdIns(3)P and micelles and forms a functionally important dimer. In addition to PtdIns(3)P-binding residues suggested by Misra and Hurley (1999), Lys-1347 is implicated in lipid recognition by NMR. The EEA1 residue Arg-1374 (which is equivalent to Arg-193 of Vps27p) is influenced similarly by PtdIns(3)P and PtdIns(5)P based on chemical shift changes and therefore is not expected to determine specificity for the 3-phosphate. EEA1’s hydrophobic element that we predict inserts into membrane is more extensive, while electrostatic membrane interactions suggested for Vps27p residue Lys-181 are not supported by micelle-specific chemical shift changes in the corresponding EEA1 residue, Lys-1362. Our direct identification of specific lipid-binding residues by NMR and

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mutational experiments provide opportunities to study the mechanism of PtdIns(3)P and membrane binding in order to gain further insight into the complex role of FYVE domains in membrane trafficking and signal transduction. Experimental Procedures

mutated products were cloned and the DNA sequences were confirmed. For random mutagenesis, a triple fusion gene, GFP-EEA1 (FYVE)-HIS3, was constructed by PCR. The FYVE domain–encoding segment of this hybrid gene was randomly mutagenized by PCR and gapped plasmid repair (Muhlrad et al., 1992). The mutant proteins were soluble, and their activities were relatively stable. Transformants were selected on media lacking histidine and screened by fluorescence microscopy for altered GFP localization.

Protein Purification A DNA fragment encoding amino acids 1325–1410 of the FYVE domain of human EEA1 was cloned into a pGEX-KG vector (Amersham Pharmacia Biotech), which was modified to eliminate all vectorencoded amino acids after thrombin cleavage except for an N-terminal GlySer dipeptide. The FYVE domain was expressed in E. coli strain BL21 pLys S grown in zinc-enriched LB media or 15NH4Cl and 13C6-glucose-supplemented M9-minimal media. The protein was purified on glutathione Sepharose 4B (Amersham Pharmacia Biotech), cleaved with thrombin (Sigma), and concentrated into 20 mM d11-Tris (pH 5 6.7 6 0.1), 200 mM KCl, 20 mM d10-DTT, 50 mM APMSF, 1 mM NaN3, in either 5% or 99.99% 2H2O/H2O.

Acknowledgments

NMR Spectroscopy NMR spectra of samples containing 0.2–2 mM unlabeled protein and uniformly 15N- and 15N/13C-labeled protein were recorded at 258C on Varian INOVA 500 MHz and 600 MHz spectrometers. Spin system and sequential assignments were made from HSQC-DIPSI, HNCO, CBCA(CO)NH, HNCACB, H(CCO)NH TOCSY, C(CO)NH TOCSY, and HCCH-TOCSY experiments collected essentially as described (Overduin et al., 1996). Interproton distances were obtained from 15 N-edited HSQC-NOESY (tm 5 50, 135, and 150 ms), HSQC-NOESYHSQC (tm 5 150 ms), and 13C-edited HMQC-NOESY spectra, which, along with 1Ha, 13Ca, 13Cb, and 13C9 chemical shifts (Wishart and Sykes, 1994) and JHNHa coupling constants derived from HNHA spectra (Vuister and Bax, 1993), were used to identify secondary structure elements. The data were analyzed using nmrPipe (Delaglio, 1993) and in-house software programs on Sun Microsystems and Silicon Graphics workstations.

References

Lipid- and Zinc-Binding Assays Lipid binding was monitored by comparing 15N-edited HSQC spectra of 0.2–0.25 mM FYVE domain in the presence of different amounts of dibutanoyl forms of PtdIns (0–0.6 mM), PtdIns(3)P (0–0.6 mM), and PtdIns(5)P (0–1 mM) or dipalmitoyl forms of these lipids (Echelon Research Laboratories) with 100 mM perdeuterated DPC (Cambridge Isotope Laboratories, Inc.) at 258C. Zinc binding was monitored from 15N-edited HSQC spectra collected immediately before and after EDTA (0, 0.1, 0.25, 0.4, 0.5, and 0.75 mM), ZnSO4 (0, 0.1, 0.25, 0.4, 0.5, and 0.75 mM), or TPEN (Sigma) was added into a FYVE domain sample (0.25 mM). Oligomeric State Diffusion coefficients were measured with pulsed field gradient NMR experiments (Altieri et al., 1995). Spectra were collected as a function of gradient steps from 0–32 G cm21 using a gradient duration time of 0.005 s and diffusion time of 0.15 s. The results were standardized against those obtained for cytochrome C under similar conditions and protein concentrations. Sedimentation velocity experiments were conducted on a Beckman XL-A instrument at 60 K rpm at 208C using a four-hole titanium rotor and two-channel charcoalfilled epon centerpieces. Data were acquired at 230 nm as one absorbance measurement per radial position at a radial spacing of 0.003 cm. Apparent sedimentation and diffusion coefficients were determined by fitting the data to the Lamm equation using the Svedberg program (Philo, 1994). Mutagenesis Site-directed mutagenesis of the EEA1 FYVE domain was done by PCR. Complementary primers designed to change codons of amino acids indicated in the text were used to amplify the FYVE domain coding sequence (amino acids 1305–1410). The products were combined, and a second PCR was done with outside primers. The

We thank R. Muhandiram and L. E. Kay for NMR pulse sequences and D. Bain for sedimentation velocity analysis. The NMR Center is supported by the Howard Hughes Medical Institute (HHMI). This research is funded by the March of Dimes and Pew Scholar’s Program (M. O.). S. D. E is supported by the HHMI and National Cancer Institute. T. G. K. and T. d. B. are the recipients of American Cancer Society and National Institutes of Health Fellowship Awards, respectively. Received May 11, 1999; revised May 27, 1999.

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