Structure of a Multifunctional Protein

MAMMALIAN PHOSPHATIDYLINOSITOL TRANSFER PROTEIN COMPLEXED WITH PHOSPHATIDYLCHOLINE*

Marilyn D. YoderDagger §, Leonard M. ThomasDagger , Jacqueline M. Tremblay||, Randall L. OliverDagger **, Lynwood R. Yarbrough||DaggerDagger, and George M. Helmkamp Jr.||DaggerDagger

From the Dagger  Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri 64110-2499 and the || Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7421

Received for publication, November 7, 2000, and in revised form, November 27, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Eukaryotic phosphatidylinositol transfer protein is a ubiquitous multifunctional protein that transports phospholipids between membrane surfaces and participates in cellular phospholipid metabolism during signal transduction and vesicular trafficking. The three-dimensional structure of the alpha -isoform of rat phosphatidylinositol transfer protein complexed with one molecule of phosphatidylcholine, one of its physiological ligands, has been determined to 2.2 Å resolution by x-ray diffraction techniques. A single beta -sheet and several long alpha -helices define an enclosed internal cavity in which a single molecule of the phospholipid is accommodated with its polar head group in the center of the protein and fatty acyl chains projected toward the surface. Other structural features suggest mechanisms by which cytosolic phosphatidylinositol transfer protein interacts with membranes for lipid exchange and associates with a variety of lipid and protein kinases.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphatidylinositol transfer proteins (PITPs)1 constitute a highly conserved family of proteins in multicellular animals, with members in species ranging from Homo sapiens to Dictyostelium discoideum (reviewed in Refs. 1-2, see also Refs. 3-5). PITPs catalyze an exchange of phosphatidylinositol (PtdIns) or phosphatidylcholine (PtdCho) molecules between the surfaces of biological or artificial membranes. The protein appears to be multifunctional, with cellular functions ranging from intermembrane phospholipid transport to possible substrate presentation to lipid-modifying enzymes. Exogenous PITP is able to restore hormone-stimulated G protein-coupled activation of PtdIns(4,5)P2 hydrolysis in human promyelocytic cells (6) and to reconstitute Ca2+-stimulated, ATP-dependent secretion of catecholamines in rat pheochromocytoma cells (7). A variety of cultured mammalian cells respond to peptide agonists, epidermal growth factor, and antigens for the IgE receptor with a PITP-dependent stimulation of PtdIns 4-kinase and phospholipase C (8-9). PITP and PtdIns 3-kinase together exhibit a capacity to phosphorylate PtdIns to PtdIns(3)P (10). In cell-free systems the budding of vesicles from the trans-Golgi network and vesicle transport between cis and medial Golgi compartments are both stimulated by PITP (11-12). Existence of PITP-containing multiprotein complexes has been proposed, poised to respond to extracellular stimuli and to channel PtdIns efficiently from PITP to kinases and phospholipases for the generation of signal transducing molecules and regulation of vesicular trafficking (13-14).

Multiple isoforms of cytosolic, 32-kDa PITPs are common and ubiquitous (15-16). Mammalian PITPalpha and PITPbeta share nearly 80% sequence identity; both isoforms are capable of forming noncovalent and stoichiometric complexes with PtdIns or PtdCho and transporting these lipid substrates through an aqueous environment. Sequence identity is more than 98% among mammalian PITPalpha isoforms (Fig. 1). PITPalpha and PITPbeta represent unique gene products (17p13.3 and 22q12.1, respectively, in humans) and they exhibit differential tissue expression. The N terminus of the 140-kDa membrane-associated retinal degeneration B proteins (rdgBs) contains a region homologous to the soluble PITPs (17-20). Significantly, the rdgB-PITP region, comprising 266 amino acids, can be expressed as a soluble protein that exhibits both PtdIns and PtdCho transfer activities. We have solved the structure of rat PITPalpha bound to phosphatidylcholine using x-ray diffraction techniques. The results provide a framework for understanding the interactions of PITPs with lipid molecules and membrane surfaces in lipid transport processes and with other cellular proteins during signaling and trafficking events.


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Fig. 1.   Sequence alignment of the PITP and rdgB proteins. Amino acids showing absolute conservation among all PITP and rdgB proteins are highlighted in red. Amino acids exhibiting 80-99% conserved identities are highlighted in yellow. Species nomenclature is as follows: RN, Rattus norvegicus; HS, H. sapien; OC, Oryctolagus cuniculus; MM, Mus musculus; DM, Drosophilia melanogaster; DD, D. discoideum; CE, C. elegans.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein Preparation and Crystallization-- There were two criteria that guided all crystallization experiments: use of unmodified protein and maintenance of a protein-bound ligand of physiological significance. Considerable biochemical data have accumulated indicating that the C-terminal residues of PITP exhibit a conformational change upon binding to membranes. We therefore avoided the use of truncated or tagged proteins, particularly at the C-terminus. To improve the likelihood that a bound phospholipid would be observed in the x-ray data, no detergents of any kind were employed in any step of purification or crystallization. Cells of Escherichia coli B834(DE3), a methionine auxotroph containing plasmids for expression of groELS and rat PITPalpha (21), were grown in chemically defined media at 20 °C with selenomethionine in place of methionine (22). The selenomethionyl-substituted protein was purified by established procedures for the wild-type protein and the bound bacterial phosphatidylglycerol (PtdGro) was replaced with sn-1,2-dioleoyl-PtdCho (21). The protein was crystallized under similar conditions as the wild-type protein when supplemented with seeding (23).

Structure Determination and Analysis-- For x-ray data collection, crystals were mounted from the crystallization drop in a fiber loop and frozen directly in a cold nitrogen stream using an ADSC cryostat. Frozen crystals were sent to NSLS beamline X12C for "Fed-Ex Data Collection" on a Brandeis B4 CCD detector. The crystal structure of the PITPalpha ·PtdCho complex was solved by multiwavelength anomalous diffraction (MAD) methods (24). A three-wavelength MAD x-ray data set was collected, with one wavelength at the selenium absorption edge, one at the absorption peak, and one at a low energy remote (Table I). Five of the eight selenium sites were identified by SOLVE (25) and refined by SHARP (26). The resulting electron density map was modified by solvent flipping as implemented in SOLOMON (27). From the resulting electron density map, the dominant secondary structure elements were traced. The final model was built through several cycles of manual model building with O (28), followed by refinement with CNS (29). The program DALI (30) was used to determine structurally related proteins. The lipid cavity was defined and volumes calculated using the CAST (31) web-server.

                              
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Table I
X-ray data collection and refinement statistics


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The protein features a large concave beta -sheet and several long alpha -helices (Fig. 2). The eight-stranded beta -sheet, strands 1 to 8, has a connectivity of 71865432. The beta -sheet is antiparallel, except for the orientation of strands 1 and 7, which are parallel. There are three long alpha -helices, composed of 5 to 7 turns (A, F, and G), and four shorter alpha -helices, each with less than two complete turns (B, C, D, and E). Helices A and F are oriented on the interior side of the beta -sheet, with helix A aligned coincident with strand 2 and helix F oriented diagonally across the beta -sheet. The phospholipid is clearly visible in the electron density map (Fig. 3), with the binding site between the interior side of the beta -sheet and alpha -helices A and F. 


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Fig. 2.   A ribbon diagram of the folding topology of PITPalpha ·PtdCho. The lipid-binding core of the protein is indicated in blue, the regulatory loop in green, and the C-terminal region in red. The phospholipid is represented in space-filled spheres using CPK coloring. beta -sheets are labeled 1-8 and alpha -helices A-G. The figure was generated with MOLSCRIPT (54) and RASTER3D (55).


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Fig. 3.   A stereo diagram of the electron density map in the vicinity of the choline group in the lipid-binding core. An omit map was calculated in which all phospholipid and water atoms were removed and one cycle of simulated annealing was run prior to the 2Fo-Fc electron density calculation. Electron density surrounding the PtdCho molecule is green, around the water molecules is light blue, and around the protein amino acids is brown. All maps are contoured at 1.0 sigma . Protein atoms are represented as thick sticks using CPK coloring and water molecules are represented by small red spheres.

Several regions of the electron density map were difficult to model. These areas consisted of residues 239-242 connecting helix F to G (containing one of the undetected selenium sites at Met241) and residues 263-271 at the C terminus. The solvent-exposed side chain density of residues on helix G is somewhat weak, although the interior, buried sidechain density is unambiguous. The solvent-exposed Met247 has very weak side chain density and is one of the undetected selenium sites from the MAD data. The last undetected selenium site is residue 1, for which no electron density is observed.

Three Functional Regions of PITP-- We designate the dominant region of the PITP structure as the lipid-binding core. This region, a cavity in which the phospholipid molecule binds, is formed by alpha -helices A and F and the beta -sheet. Limited segments of helices A and F and strands 4 and 5 are amphipathic and provide excellent hydrophobic interaction with the phospolipid molecule (Fig. 4). The lipid-binding core comprises residues 1-118 and 191-232. Examination of the PITP structure reveals that the intervening residues 119-190 form an extensive loop region between strands 7 and 8. Postulating that this loop is likely required for the association of PITP with a wide variety of lipid- and protein-modifying enzymes, we refer to this region as the regulatory loop. The recent identification of Ser166 as a site for limited protein kinase C-dependent phosphorylation (32) supports this hypothesis. All PITPs and rdgBs studied to date contain a regulatory loop region; only Caenorhabditis elegans PITP lacks the equivalent Ser166 phosphorylation site. The remaining residues in the structure comprise the C-terminal residues 233-271. Consisting of the long alpha -helix G as the principal secondary structural element and an additional 11 residues that limit access to the lipid-binding cavity, we call this region the C-terminal region and suggest its critical participation in membrane association and dissociation. The C-terminal region is the least conserved region among the cytosolic PITPs and is replaced in the rdgB proteins by up to 1000 additional amino acids, some of which appear to be transmembrane.


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Fig. 4.   The phospholipid-binding pocket. Residues and water molecules within 3.9 Å of the PtdCho ligand are indicated. Hydrogen bonds with the polar head group are indicated by dashed lines. Water molecules are indicated by small spheres. The sn-1 acyl chain is on the right, and the sn-2 acyl chain is on the left. The figure was generated with LIGPLOT (56).

Our functional region designation correlates reasonably well to the location of conserved amino acid residues in the PITP family (Fig. 1). The majority (nearly 75%) of the absolutely conserved amino acids are located in the lipid-binding core. Most of the others are part of the regulatory loop. Consistent with their proposed functions, both the regulatory loop and the C-terminal region were predicted to exhibit significant polypeptide backbone flexibility (33), a structural feature corroborated by specific B factors from the x-ray data.

Characterization of the Lipid-binding Cavity-- The residues in close proximity to the PtdCho head group are shown in Figs. 3 and 4. The majority of these interacting residues are highly conserved in all PITP and rdgB proteins. Nine water molecules are within 3.2 Å of the phospholipid or an amino acid residue lining the phospholipid cavity. The phosphate oxygen O1P forms hydrogen bonds with the NZ atom of the conserved Lys195 and a water molecule, and oxygen O2P hydrogen bonds with the OG1 atom of the conserved Thr97 and another water molecule. The solvent-accessible volume of the phospholipid-binding cavity (calculated with a 1.4 Å probe) is 670 Å3, whereas the molecular-surface volume is 2725 Å3. The van der Waals volume of the PtdCho molecule is 671 Å3. Of the 271 residues in the protein, 72 residues (27%) have atoms contributing to the lipid cavity.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characteristics of the interactions between PITP and the hydrophobic region of PtdIns and PtdCho molecules have been described (34-35). Using lipids with defined fatty acyl species at their sn-1 and sn-2 positions, combined with one or more fluorescent acyl derivatives (pyrenylacyl, parinaroyl), both binding to PITP and protein-catalyzed transfer from a vesicle surface were measured. For the sn-1 group a broad range of structures could be accommodated, with preference for 14- and 16-carbon saturated or monounsaturated fatty acids; nevertheless, 18-carbon polyunsaturated fatty acids were still bound and transferred. In contrast, the sn-2 group exhibits a more restricted range, preferring 16- and 18-carbon mono- and polyunsaturated fatty acids whose double bonds were toward the methyl-end of the lipids. PtdCho molecules containing arachidonate (20:4n-6) or docosahexaenoate (22:6n-3) in the sn-2 position bind weakly to PITP. It was suggested that the sn-2 binding site in PITP was "bottle-shaped," with the neck toward the ester linkage. Little difference was observed for acyl chain preferences between the PtdIns and PtdCho class of phospholipids.

In the PITP structure reported here, the PtdCho ligand contains two oleate (18:1n-9) acyl groups. The conformation of the sn-2 acyl chain is more extended than the sn-1 chain, such that the sn-2 group extends almost to the surface of the protein. The lipid cavity follows the contour of the sn-2 acyl chain closely, but the cavity around the sn-1 chain is less confining. These features may explain the poor binding of phospholipid molecules with sn-2 acyl moieties longer than 18 carbons and the less restrictive range of groups accommodated in the sn-1 acyl region of the cavity.

PITP preferentially binds PtdIns and PtdCho. Whereas PtdCho was used in the experimental protocol, inositol can be readily modeled into the electron density for the choline moiety (except for the sugar hydroxyls). There are numerous charged or polar amino acid residues in the region that may be potential hydrogen bonding partners to the inositol hydroxyls (Fig. 5). Nevertheless, the tertiary structure of the model does not completely explain polar head group specificity of PITP, for which further studies are needed.


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Fig. 5.   PtdIns modeled into the electron density map of PITPalpha ·PtdCho. Only electron density from the final 2Fo-Fc map surrounding the PtdCho is shown.

Conformation, Stability, and Function of the C-terminal Region-- To transfer a phospholipid, PITP must first bind to the membrane, exchange its bound lipid for a membrane lipid, and then dissociate from the membrane. Previous studies have shown that there are significant conformational changes in the C-terminal residues when PITP binds to lipid membranes (21). Although cytosolic PITP is highly resistant to trypsin digestion, protein bound to membranes is rapidly cleaved at Arg253 and Arg259. In the crystal structure these two sites are part of helix G in the C-terminal region. Studies have shown that generally sites that are subject to limited proteolytic cleavage by trypsin assume a loop-like structure with a conformation similar to that of trypsin inhibitors (36). Thus, this helix must "melt" prior to cleavage by trypsin, a process facilitated by interaction with a membrane surface. The relative instability of portions of this helix is supported by the high B factors calculated for residues 243-250.

Residues 260-271 also appear to be important for maintaining a tight compact structure. PITPs truncated at residues 253 or 259 have more relaxed conformations than full-length PITP, defined by increased quenching of Trp fluorescence by acrylamide, increased Tyr exposure, increased reactivity of Cys, and increased anilino-naphthalene sulfonate binding (37). Inspection of the native structure shows that such changes cannot be caused by removal and exposure of residues without a conformational change in the remainder of the protein. Several studies have shown that removal of one or a few amino acids from the N- or C-terminus of a protein may have a significant effect on protein stability and/or dynamics without producing major changes in conformation (38-39). Our data on the conformation and biological activity of truncated PITPs are consistent with a critical role of the C-terminus in the complete packing of the hydrophobic core and folding and stabilization of the native structure.

The ability of PITP to bind phospholipid substrates is necessary but not sufficient for subsequent phospholipid transfer. Removal of up to 20 C-terminal residues greatly reduces phospholipid transfer, yet these truncated proteins produced in E. coli contain PtdGro, or less commonly, phosphatidylethanolamine (PtdEtn) with a near normal 1:1 stoichiometry (37, 40). Hence, residues 254-271 are dispensable for lipid binding, although they are important in conferring selectivity for PtdIns and for efficient transfer of phospholipids. It is not immediately obvious from the PITP structure why phospholipids such as PtdEtn and phosphatidic acid (PtdOH) are bound and/or transferred so poorly.

We had earlier reported that residues 260-271 could be eliminated with variable effects on transfer activity that reflected the composition of the membranes employed in the assay system (41). With membranes containing only PtdCho, the truncated PITP (1) has transfer activity identical to the full-length protein. However, with as little as 2% PtdOH in the donor and/or acceptor membranes, transfer activity decreases to 0-20% relative to that observed with PtdCho membranes. Such concentrations have minimal effect on transfer by native PITP. We demonstrated that this and the more extensively truncated PITP (1) have greatly enhanced affinities for membrane binding, which would result in inefficient lipid transfer activity. We suggest that the C-terminal region of PITP acts to maintain a low affinity of the full-length protein for membranes, especially those containing acidic phospholipids. Structural perturbations within the C-terminal region lead to enhanced electrostatic interactions and increased membrane affinity (41).

Structure-Function Correlation of Point Mutations-- Knowledge of the tertiary structure of PITP provides an opportunity to evaluate studies describing and characterizing the random mutagenesis of the cDNA encoding rat PITPalpha (42). Mutations of amino acids Ser25, Thr59, Pro78, and Glu248 were, in general, observed to abolish PtdIns transfer, whereas PtdCho transfer was not affected. With the exception of Ser25, all are absolutely conserved among PITPs and rdgBs. Our data support a role of the Thr59 hydroxyl group in a putative hydrogen bonding interaction with the inositol moiety (Fig. 5) and place Ser25 in the vicinity of the fatty acid-glycerol ester linkages. Glu248 makes a salt bridge with the conserved Lys61 in a region close to the choline and inositol polar head group moieties. Pro78 is near the end of the portion of the cavity that accommodates the sn-2 acyl chain. Why mutations of these residues impact selectively and variably on PtdIns binding and transfer remains unresolved.

Mutations to Glu6 and Pro12, two absolutely conserved residues, yield poor expression of recombinant mammalian PITP in a yeast host (42). Based on our structure, these residues likely stabilize the association between the lipid-binding core and the regulatory loop. Glu6 participates in salt bridges with the conserved Arg8 and with His127; similarly, Pro12 packs tightly against the conserved Leu174.

Membrane Binding and Mechanism of Phospholipid Transfer-- Analysis of the exterior features of PITP, such as charge distribution and hydrophobicity, provided little insight into what region might constitute the membrane-binding surface. We presently favor a model in which the C-terminal region is a part of this surface. In the interaction between protein and membrane, most if not all of the C-terminal region must be displaced from helix A in the lipid-binding core and acquire increased backbone flexibility sufficient to melt a portion of helix G and become sensitive to proteolytic digestion. We further envision that the refolding and reassociation of the C-terminal region with the bulk protein provides a driving force for eventual dissociation of PITP from the membrane surface.

The detailed mechanism by which PITP releases its bound lipid, exchanges it for another present in the membrane surface and dissociates from the membrane remains to be elucidated. A plausible model is one in which the C-terminal region of the protein binds to the membrane and the lipid-binding core opens, somewhat like a clam shell, exposing its bound lipid to the membrane surface. The protein-bound lipid is absorbed into the membrane and replaced by a membrane lipid. Subsequently, the C-terminal region refolds and reassociates with the body of the protein, i.e. the clam shell closes, and the protein dissociates from the membrane surface. The affinity for the membrane surface and the affinity for a specific phospholipid must not be too great, otherwise dissociation and transfer would be too slow and inefficient. As suggested above, the decreased rate of membrane dissociation, at least in part, makes C-terminal-truncated PITP derivatives weak catalysts of lipid exchange with membranes containing acidic phospholipids.

To account for other biological activities of PITP, we suggest that the protein-bound phospholipid becomes accessible to phosphorylation following interaction of a lipid kinase with a complementary site on PITP, perhaps on the regulatory loop. One-third of the residues within this 72-amino acid segment are identical or >90% conserved among all PITP and rdgB proteins (Fig. 1). Protein-protein interactions are proposed to alter the conformation of PITP at the head group region of the lipid-binding core (Fig. 6). This could lead to either the presentation of PtdIns to the active site of a membrane-bound PtdIns 4-kinase or the exposure of the inositol moiety of a PITP-bound PtdIns to the action of a soluble PtdIns 3-kinase or 4-kinase (43-44). Evidence supporting such a function of PITP in substrate presentation to a PtdIns 3-kinase has been reported (10). Following modification of the inositol 3- or 4-hydroxyl function, the affinity of the product for PITP would likely decrease so significantly that it would remain with the kinase or return to the lipid surface. Consistent with this mechanism is the inability of PITP to bind and transfer PtdIns (4)P (45).


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Fig. 6.   Schematic of interaction of PITP with membrane surfaces and other proteins. A, protein-catalyzed phospholipid exchange and transport between membranes. B, interaction of a membrane-bound PITP and cytosolic PtdIns 3-kinase. C, interaction of PITP with a membrane-bound PtdIns 4-kinase.

Comparison with other Lipid-binding Proteins-- Using the DALI algorithm (30) to search for proteins structurally similar to PITP, the recently described START (StAR-related lipid-transfer) domain (46) was identified as sharing significant tertiary structure. MLN64 (PDB:1EM2), a protein associated with some human breast carcinomas, has served as a prototype for structure determination and mechanistic studies of the START domain (47). The MLN64 START domain contains a hydrophobic tunnel that extends the length of the protein with small openings on both ends. A nine-stranded antiparallel beta -sheet and two alpha -helices form an interior hydrophobic chamber that is believed to be just large enough to accommodate a single molecule of cholesterol. Cholesterol was not included in the crystallization solutions, but docked into the finished protein model. START domain proteins are proposed to bind lipids and interact with membranes, properties also common to PITPs. Steroidogenic acute regulatory (StAR) protein, whose function is to deliver cholesterol to the inner mitochondrial membrane and cytochrome P450scc, contains a START domain near its C-terminus. Nearly the entire sequence of PtdCho transfer protein (PCTP), another cytosolic lipid transfer protein, constitutes a single START domain. In contrast to PITP, PCTP has a marked specificity for PtdCho and highly restricted tissue expression in mammals (2). Although not identified by the DALI search, the recently reported structure of sterol carrier protein-2 (PDB 1QND, Ref. 48) is another example of a protein with a lipid-binding core similar to that observed in MLN64 and PITP.

Despite reasonable similarity at the secondary and tertiary levels of structure (Fig. 7), there is no primary structure homology between members of the PITP family and proteins with a START domain. It should be noted that the lipid cavity in PITP is larger than the cavity in MLN64. Upon superposition, one of the alpha -helices comprising the putative lipid-binding cavity of MLN64 clashes with the sn-1 acyl chain of the bound PtdCho in the PITP structure. This is not surprising in that PITP binds diacyl phospholipids, whereas MLN64 binds the more compact cholesterol molecule. The START and PITP protein families represent an excellent example of functional evolutionary convergence to generate comparable domain architecture and related biological function from apparently unrelated genetic information.


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Fig. 7.   Stereo diagram of the superposition of the Calpha atoms of the PITPalpha ·PtdCho lipid-binding core and the START domain of MLN64. The PITP trace is blue, and the MLN64 trace is red. The PtdCho molecule of PITPalpha ·PtdCho is shown in sticks.

Fungi and plants contain Sec14p, a cytosolic protein that transports PtdIns and PtdCho (13, 49-52) and is required for protein export and cell viability. Recently, the three-dimensional structure of Saccharomyces cerevisiae Sec14p, one member of this conserved family, was reported (53). PITP and Sec14p are similar in size, exhibit similar in vitro lipid-binding specificity and transport abilities and display significant and highly complementary interspecies activity. However, the two proteins have little structural similarity. In contrast to the PITPalpha ·PtdCho structure, Sec14p structure has no bound phospholipid molecule.

Conclusions-- Elucidation of the PITPalpha ·PtdCho structure reveals a complex amphipathic lipid completely enclosed within a folded polypeptide cavity. The three functional regions of the protein provides a molecular framework for understanding the details of protein-lipid interactions on several levels. Within its lipid-binding core, PITP has been shown to sequester phospholipids from the aqueous environment. Lipid ligand specificity, whereas not completely explained by the tertiary structure, is dictated by extensive noncovalent interactions. The C-terminal region of PITP is proposed to promote the transient association to and dissociation from lipid surfaces. Through its regulatory loop, PITP is thought to present lipid substrates to kinases for subsequent phosphorylation. Further mutagenic and structural studies of PITP and other members of the PITP-rdgB family are required to test these and other hypotheses. Detailed analyses of PITP interactions with membranes and kinases are needed to gain a more meaningful understanding of the role of PITP in cellular phospholipid metabolism.

    ACKNOWLEDGEMENTS

The atomic coordinates for the crystal structure of PITPalpha ·PtdCho have been deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank with accession code 1FVZ. We thank Chuong Doan and Sienna Sifuentes for technical assistance. Diffraction data for this study were collected at Brookhaven National Laboratory in the Biology Department single-crystal diffraction facility at beamline X12-C in the National Synchrotron Light Source. This facility is supported by the United States Department of Energy Offices of Health and Environmental Research and of Basic Energy Sciences under prime contract DE-AC02-98CH10886, by the National Science Foundation and by National Institutes of Health Grant 1P41 RR12408-01A1. We are grateful for the assistance of Michael Becker and Robert Sweet with "Fed-Ex Data Collection" at X12-C.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM24035 (to G. M. H. and L. R. Y.) and GM59162 (to G. M. H., L. R. Y., and M. D. Y.), the American Heart Association (to L. R. Y.), and the University of Kansas Medical Center Research Institute (to G. M. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1FVZ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

§ To whom correspondence should be addressed: Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 5100 Rockhill Rd., Kansas City, MO 64110-2499. Tel.: 816-235-1986; Fax: 816-235-1503; E-mail: yoderm@umkc.edu.

Present address: Howard Hughes Medical Inst., Division of Biology, California Inst. of Technology, Pasadena, CA 91125.

** Present address: Zymogenetics, 1201 Eastlake Ave. E., Seattle, WA 98102.

Dagger Dagger These authors contributed equally to this work.

Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M010131200

    ABBREVIATIONS

The abbreviations used are: PITP, phosphatidylinositol transfer protein; PtdIns, phosphatidylinositol; PtdCho, phosphatidylcholine; rdgB, retinol degeneration B protein; PtdGro, phosphatidylglycerol; MAD, multiwavelength anomalous diffraction; PtdEtn, phosphatidylethanolamine; PtdOH, phosphatidic acid; START, StAR-related lipid-transfer; StAR, steroidogenic acute regulatory; PCTP, phosphatidylcholine transfer protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Trotter, P. J., and Voelker, D. R. (1994) Biochim. Biophys. Acta 1213, 241-262[Medline] [Order article via Infotrieve]
2. Wirtz, K. W. A. (1997) Biochem. J. 324, 353-360[Medline] [Order article via Infotrieve]
3. Lev, S., Hernandez, J., Martinez, R., Chen, A., Plowman, G., and Schlessinger, J. (1999) Mol. Cell. Biol. 19, 2278-2288[Abstract/Free Full Text]
4. Swigert, P., Insall, R., Wilkins, A., and Cockcroft, S. (2000) Biochem. J. 347, 837-843[CrossRef][Medline] [Order article via Infotrieve]
5. The C. elegans sequencing consortium. (1998) Science 282, 2012-2018[Abstract/Free Full Text]
6. Thomas, G. M. H., Cunningham, E., Fensome, A., Ball, A., Totty, N. F., Truong, O., Hsuan, J. J., and Cockcroft, S. (1993) Cell 74, 919-928[Medline] [Order article via Infotrieve]
7. Hay, J. C., and Martin, T. F. J. (1993) Nature 366, 572-575[CrossRef][Medline] [Order article via Infotrieve]
8. Kauffmann-Zeh, A., Thomas, G. M. H., Ball, A., Prosser, S., Cunningham, E., Cockcroft, S., and Hsuan, J. J. (1995) Science 268, 1188-1190[Medline] [Order article via Infotrieve]
9. Cunningham, E., Tan, S. K., Swigert, P., Hsuan, J., Bankaitis, V., and Cockcroft, S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6589-6593[Abstract/Free Full Text]
10. Panaretou, C., Domin, J., Cockcroft, S., and Waterman, M. D. (1997) J. Biol. Chem. 272, 2477-2485[Abstract/Free Full Text]
11. Ohashi, M., de Vries, K. J., Frank, R., Snoek, G., Bankaitis, V., Wirtz, K., and Huttner, W. B. (1995) Nature 377, 544-547[CrossRef][Medline] [Order article via Infotrieve]
12. Paul, K. S., Bogan, A. A., and Waters, M. G. (1998) FEBS Lett. 431, 91-96[CrossRef][Medline] [Order article via Infotrieve]
13. Cockcroft, S. (1998) BioEssays 20, 423-432[CrossRef][Medline] [Order article via Infotrieve]
14. Martin, T. F. J. (1997) Curr. Opin. Neurobiol. 7, 331-338[CrossRef][Medline] [Order article via Infotrieve]
15. Tanaka, S., and Hosaka, K. (1994) J. Biochem. (Tokyo) 115, 981-984[Abstract]
16. Dickeson, S. K., Lim, C. N., Schuyler, G. T., Dalton, T. P., Helmkamp, G. M., Jr., and Yarbrough, L. R. (1989) J. Biol. Chem. 264, 16557-16564[Abstract/Free Full Text]
17. Vihtelic, T. S., Goebl, M., Milligan, S., O'Tousa, J. E., and Hyde, D. R. (1993) J. Cell Biol. 122, 1013-1022[Abstract]
18. Guo, J., and Yu, F. X. (1997) Dev. Genet. 20, 235-245[CrossRef][Medline] [Order article via Infotrieve]
19. Chang, J. T., Milligan, S., Li, Y., Chew, C. E., Wiggs, J., Copeland, N. G., Jenkins, N. A., Campochiaro, P. A., Hyde, D. R., and Zack, D. J. (1997) J. Neurosci. 17, 5881-5890[Abstract/Free Full Text]
20. Aikawa, Y., Hara, H., and Watanabe, T. (1997) Biochem. Biophys. Res. Comm. 236, 559-565[CrossRef][Medline] [Order article via Infotrieve]
21. Tremblay, J. M., Helmkamp, G. M., Jr., and Yarbrough, L. R. (1996) J. Biol. Chem. 271, 21075-21080[Abstract/Free Full Text]
22. Neidhardt, F. C., Bloch, P. L., and Smith, D. F. (1975) J. Bacteriol. 119, 736-747
23. Oliver, R. L., Tremblay, J. M., Helmkamp, G. M., Jr., Yarbrough, L. R., Breakfield, N. W., and Yoder, M. D. (1999) Acta Crystallogr. Sect. D 55, 522-524[CrossRef][Medline] [Order article via Infotrieve]
24. Hendrickson, W. A. (1991) Science 254, 51-58[Medline] [Order article via Infotrieve]
25. Terwilliger, T. C., and Berendzen, J. (1999) Acta Crystallogr. Sect. D 55, 849-861[CrossRef][Medline] [Order article via Infotrieve]
26. La Fortelle, E., and Bricogne, G. (1997) Methods Enzymol. 276, 472-494
27. Abrahams, J. P., and Leslie, A. G. W. (1996) Acta Crystallogr. Sect. D 52, 32-42[CrossRef]
28. Jones, T. A., Cowan, S., Zou, J.-Y., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve]
29. Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J.-S., Kuszewski, J., Nilges, N., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
30. Holm, L., and Sander, C. (1993) J. Mol. Biol. 233, 123-138[CrossRef][Medline] [Order article via Infotrieve]
31. Liang, J., Edelsbrunner, H., and Woodward, C. (1998) Prot. Sci. 7, 1884-1897[Abstract/Free Full Text]
32. van Tiel, C. M., Westerman, J., Paasman, M., Wirtz, K. W. A., and Snoek, G. T. (2000) J. Biol. Chem. 275, 21532-21538[Abstract/Free Full Text]
33. Karplus, P. A., and Schultz, G. E. (1985) Naturwissenschaften 72, 212-213
34. van Paridon, P. A., Gadella, T. W. J., Jr., Somerharju, P. J., and Wirtz, K. W. A. (1988) Biochemistry 27, 6208-6214[Medline] [Order article via Infotrieve]
35. Kasurinen, J., van Paridon, P. A., Wirtz, K. W. A., and Somerharju, P. (1990) Biochemistry 29, 8548-8554[Medline] [Order article via Infotrieve]
36. Hubbard, S. J., Eisenmenger, F., and Thornton, J. M. (1994) Prot. Sci. 3, 757-768[Abstract/Free Full Text]
37. Voziyan, P. A., Tremblay, J. M., Yarbrough, L. R., and Helmkamp, G. M., Jr. (1996) Biochemistry 35, 12526-12531[CrossRef][Medline] [Order article via Infotrieve]
38. Trevino, R. J., Gliubich, F., Berni, R., Cianci, M., Chirgwin, J. M., Zanotti, G., and Horowitz, P. M. (1999) J. Biol. Chem. 274, 13938-13947[Abstract/Free Full Text]
39. de Prat Gay, G., Ruiz-Sanz, J., Neira, J. L., Corrales, F. J., Otzen, D. E., Ladurner, A. G., and Fersht, A. R. (1995) J. Mol. Biol. 254, 968-979[CrossRef][Medline] [Order article via Infotrieve]
40. Hara, S., Swigart, P., Jones, D., and Cockcroft, S. (1997) J. Biol. Chem. 272, 14908-14913[Abstract/Free Full Text]
41. Tremblay, J. M., Voziyan, P. A., Helmkamp, G. M., Jr., and Yarbrough, L. R. (1998) Biochim. Biophys. Acta 1389, 91-100[Medline] [Order article via Infotrieve]
42. Alb, J. G., Jr., Gedvilaite, A., Cartee, R. T., Skinner, H. B., and Bankaitis, V. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8826-8830[Abstract]
43. Balla, T. (1998) Biochim. Biophys. Acta 1436, 69-85[Medline] [Order article via Infotrieve]
44. Wymann, M. P., and Pirola, L. (1998) Biochim. Biophys. Acta 1436, 127-150[Medline] [Order article via Infotrieve]
45. Schermoly, M. J., and Helmkamp, G. M., Jr. (1983) Brain Res. 268, 197-200[Medline] [Order article via Infotrieve]
46. Ponting, C. P., and Aravind, L. (1999) Trends Biochem. Sci. 24, 130-132[CrossRef][Medline] [Order article via Infotrieve]
47. Tsujishita, Y., and Hurley, J. H. (2000) Nat. Struct. Biol. 7, 408-414[CrossRef][Medline] [Order article via Infotrieve]
48. Garcia, F. L., Szyperski, T., Dyer, J. H., Choinowski, T., Seedorf, U., Hauser, H., and Wüthrich, K. (2000) J. Mol. Biol. 295, 595-603[CrossRef][Medline] [Order article via Infotrieve]
49. Kearns, B. G., Alb, J. G., Jr., and Bankaitis, V. A. (1998) Trends Cell Biol. 8, 276-282[CrossRef][Medline] [Order article via Infotrieve]
50. Bankaitis, V. A., Aitken, J. R., Cleves, A. E., and Dowhan, W. (1990) Nature 347, 561-562[CrossRef][Medline] [Order article via Infotrieve]
51. Daum, G., and Paltauf, F. (1984) Biochim. Biophys. Acta 794, 385-391
52. Jouannic, N., Lepetit, M., Vergnolle, C., Cantrel, C., Gardies, A.-M., Kader, J.-C., and Arondel, V. (1998) Eur. J. Biochem. 258, 402-410[Abstract]
53. Sha, B., Phillips, S. E., Bankaitis, V. A., and Luo, M. (1998) Nature 391, 506-510[CrossRef][Medline] [Order article via Infotrieve]
54. Kraulis, P. J. (1991) J. App. Crystallogr. 24, 946-950[CrossRef]
55. Merritt, E. A., and Murphy, M. E. P. (1994) Acta Crystallogr. Sect. D 50, 869-873[CrossRef][Medline] [Order article via Infotrieve]
56. Wallace, A. C., Laskowski, R. A., and Thornton, J. M. (1995) Prot. Eng. 8, 127-134[Abstract]


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