Structure of a Multifunctional Protein

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 α-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 β-sheet and several long α-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.1FVZ 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 α-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 β-sheet and several long α-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. 1FVZ phosphatidylinositol transfer protein phosphatidylinositol phosphatidylcholine retinol degeneration B protein phosphatidylglycerol multiwavelength anomalous diffraction phosphatidylethanolamine phosphatidic acid StAR-related lipid-transfer steroidogenic acute regulatory phosphatidylcholine transfer protein 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. 1Trotter P.J. Voelker D.R. Biochim. Biophys. Acta. 1994; 1213: 241-262Crossref PubMed Scopus (87) Google Scholar, 2Wirtz K.W.A. Biochem. J. 1997; 324: 353-360Crossref PubMed Scopus (174) Google Scholar, see also Refs. 3Lev S. Hernandez J. Martinez R. Chen A. Plowman G. Schlessinger J. Mol. Cell. Biol. 1999; 19: 2278-2288Crossref PubMed Google Scholar, 4Swigert P. Insall R. Wilkins A. Cockcroft S. Biochem. J. 2000; 347: 837-843Crossref PubMed Google Scholar, 5The C. elegans sequencing consortium, Science, 282, 1998, 2012, 2018.Google Scholar). 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 (6Thomas G.M.H. Cunningham E. Fensome A. Ball A. Totty N.F. Truong O. Hsuan J.J. Cockcroft S. Cell. 1993; 74: 919-928Abstract Full Text PDF PubMed Scopus (188) Google Scholar) and to reconstitute Ca2+-stimulated, ATP-dependent secretion of catecholamines in rat pheochromocytoma cells (7Hay J.C. Martin T.F.J. Nature. 1993; 366: 572-575Crossref PubMed Scopus (306) Google Scholar). 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 (8Kauffmann-Zeh A. Thomas G.M.H. Ball A. Prosser S. Cunningham E. Cockcroft S. Hsuan J.J. Science. 1995; 268: 1188-1190Crossref PubMed Scopus (160) Google Scholar, 9Cunningham E. Tan S.K. Swigert P. Hsuan J. Bankaitis V. Cockcroft S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6589-6593Crossref PubMed Scopus (99) Google Scholar). PITP and PtdIns 3-kinase together exhibit a capacity to phosphorylate PtdIns to PtdIns(3)P (10Panaretou C. Domin J. Cockcroft S. Waterman M.D. J. Biol. Chem. 1997; 272: 2477-2485Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). In cell-free systems the budding of vesicles from thetrans-Golgi network and vesicle transport betweencis and medial Golgi compartments are both stimulated by PITP (11Ohashi M. de Vries K.J. Frank R. Snoek G. Bankaitis V. Wirtz K. Huttner W.B. Nature. 1995; 377: 544-547Crossref PubMed Scopus (168) Google Scholar, 12Paul K.S. Bogan A.A. Waters M.G. FEBS Lett. 1998; 431: 91-96Crossref PubMed Scopus (18) Google Scholar). 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 (13Cockcroft S. BioEssays. 1998; 20: 423-432Crossref PubMed Scopus (86) Google Scholar, 14Martin T.F.J. Curr. Opin. Neurobiol. 1997; 7: 331-338Crossref PubMed Scopus (84) Google Scholar). Multiple isoforms of cytosolic, 32-kDa PITPs are common and ubiquitous (15Tanaka S. Hosaka K. J. Biochem. ( Tokyo ). 1994; 115: 981-984Crossref PubMed Scopus (84) Google Scholar, 16Dickeson S.K. Lim C.N. Schuyler G.T. Dalton T.P. Helmkamp Jr., G.M. Yarbrough L.R. J. Biol. Chem. 1989; 264: 16557-16564Abstract Full Text PDF PubMed Google Scholar). Mammalian PITPα and PITPβ 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 PITPα isoforms (Fig.1). PITPα and PITPβ 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 (17Vihtelic T.S. Goebl M. Milligan S. O'Tousa J.E. Hyde D.R. J. Cell Biol. 1993; 122: 1013-1022Crossref PubMed Scopus (160) Google Scholar, 18Guo J. Yu F.X. Dev. Genet. 1997; 20: 235-245Crossref PubMed Scopus (30) Google Scholar, 19Chang J.T. Milligan S. Li Y. Chew C.E. Wiggs J. Copeland N.G. Jenkins N.A. Campochiaro P.A. Hyde D.R. Zack D.J. J. Neurosci. 1997; 17: 5881-5890Crossref PubMed Google Scholar, 20Aikawa Y. Hara H. Watanabe T. Biochem. Biophys. Res. Comm. 1997; 236: 559-565Crossref PubMed Scopus (38) Google Scholar). 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 PITPα 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. 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 coliB834(DE3), a methionine auxotroph containing plasmids for expression of groELS and rat PITPα (21Tremblay J.M. Helmkamp Jr., G.M. Yarbrough L.R. J. Biol. Chem. 1996; 271: 21075-21080Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), were grown in chemically defined media at 20 °C with selenomethionine in place of methionine (22Neidhardt F.C. Bloch P.L. Smith D.F. J. Bacteriol. 1975; 119: 736-747Crossref Google Scholar). The selenomethionyl-substituted protein was purified by established procedures for the wild-type protein and the bound bacterial phosphatidylglycerol (PtdGro) was replaced withsn-1,2-dioleoyl-PtdCho (21Tremblay J.M. Helmkamp Jr., G.M. Yarbrough L.R. J. Biol. Chem. 1996; 271: 21075-21080Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The protein was crystallized under similar conditions as the wild-type protein when supplemented with seeding (23Oliver R.L. Tremblay J.M. Helmkamp Jr., G.M. Yarbrough L.R. Breakfield N.W. Yoder M.D. Acta Crystallogr. Sect. D. 1999; 55: 522-524Crossref PubMed Scopus (5) Google Scholar). 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 PITPα·PtdCho complex was solved by multiwavelength anomalous diffraction (MAD) methods (24Hendrickson W.A. Science. 1991; 254: 51-58Crossref PubMed Scopus (1015) Google Scholar). 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 (TableI). Five of the eight selenium sites were identified by SOLVE (25Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D. 1999; 55: 849-861Crossref PubMed Scopus (3219) Google Scholar) and refined by SHARP (26La Fortelle E. Bricogne G. Methods Enzymol. 1997; 276: 472-494Crossref PubMed Scopus (1797) Google Scholar). The resulting electron density map was modified by solvent flipping as implemented in SOLOMON (27Abrahams J.P. Leslie A.G.W. Acta Crystallogr. Sect. D. 1996; 52: 32-42Crossref Scopus (1142) Google Scholar). 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 (28Jones T.A. Cowan S. Zou J.-Y. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13009) Google Scholar), followed by refinement with CNS (29Brünger 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. Warren G.L. Acta Crystallogr. Sect. D. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar). The program DALI (30Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3561) Google Scholar) was used to determine structurally related proteins. The lipid cavity was defined and volumes calculated using the CAST (31Liang J. Edelsbrunner H. Woodward C. Prot. Sci. 1998; 7: 1884-1897Crossref PubMed Scopus (866) Google Scholar) web-server.Table IX-ray data collection and refinement statisticsData collectionSpace group, Unit cellP21 a = 43.914 Å, b = 73.773 Å, c = 48.185 Å, β = 114.732°BeamlineNSLS, X12-CResolution2.2 ÅData setWave-lengthNo. observationsNo. unique reflectionsCompleteness 1-aValues in parentheses indicate the highest resolution shell.I/ςMosaicityRsym 1-bRsym = Σ‖I(hkl) − 〈I(hkl)〉‖/ΣI(hkl).Å%degree%Peak0.979141,31413,08190.1 (67.2)20.2 (7.2)0.6414.5 (8.1)Edge0.979439,67112,87987.4 (60.8)18.0 (6.2)0.6405.1 (9.3)Remote0.950037,69012,66987.8 (64.0)18.4 (6.4)0.6244.6 (8.6)RefinementResolution range (Å)50.0–2.2Rcryst(%)1-cRcryst = Σ‖Fobs(hkl) −Fcalc(hkl)‖/ΣFobs(hkl).21.2Rfree (%)25.3Number of reflections 1-dAll data included in calculations of Working set and Test set.Working set11,516 (80.8%)Test set629 (4.4%)Number of atomsProtein (residues 2–270)2,228Lipid (1 molecule from and from in parentheses indicate the highest resolution = Σ‖I(hkl) − = Σ‖Fobs(hkl) data included in calculations of Working set and Test and from in a The protein features a β-sheet and several long α-helices The to has a of The β-sheet is for the of and which are There are long of to and and with than two and A and are on the of the with A with and the The phospholipid is in the electron density map with the binding between the of the β-sheet and α-helices A and of the electron density map in the of the group in the map was calculated in which all phospholipid and were and one of was to the electron density density the PtdCho molecule is the molecules and the protein amino are at are as using and molecules are by of the electron density map were to of residues to G one of the selenium sites at and residues at the C The density of residues on G is the density is The has density and is one of the selenium sites from the MAD The selenium is for which no electron density is We the dominant region of the PITP structure as the region, a cavity in which the phospholipid molecule is by α-helices A and and the of A and and and are and provide with the molecule The residues and of the PITP structure that the residues an loop region between and that loop is for the of PITP with a variety of and to region as the regulatory The of as a for protein J. M. Wirtz K.W.A. Snoek G.T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) PITPs and to a regulatory loop elegans PITP the The residues in the structure the C-terminal residues of the long G as the secondary structural and an residues that to the region the C-terminal region and suggest its in membrane and The C-terminal region is the conserved region among the cytosolic PITPs and is replaced in the proteins by to amino acids, of which to be region to the of conserved amino acid residues in the PITP family The of the conserved amino are in the of the are of the regulatory with both the regulatory loop and the C-terminal region were to exhibit P.A. Scopus Google Scholar), a structural by B from the x-ray The residues in to the PtdCho head group are in The of these residues are highly conserved in all PITP and proteins. molecules are Å of the phospholipid or an amino acid the phospholipid The with the of the conserved and a and with the of the conserved and The of the cavity with a Å is the is The of the PtdCho molecule is the residues in the residues have to the lipid of the interactions between PITP and the region of PtdIns and PtdCho molecules have been P.A. Jr., P.J. Wirtz K.W.A. PubMed Scopus Google Scholar, J. P.A. Wirtz K.W.A. P. PubMed Scopus Google Scholar). with defined fatty acyl species at and with one or more acyl both binding to PITP and transfer from a vesicle were For the group a range of be with for and or fatty fatty were bound and In the group exhibits a more and and fatty were toward the of the PtdCho molecules containing or in the to was that the binding in PITP was with the toward the was observed for acyl between the PtdIns and PtdCho of In the PITP structure the PtdCho ligand contains two acyl The of the acyl is more than the that the group to the of the The lipid cavity the of the acyl the cavity the is features the binding of phospholipid molecules with acyl than and the range of accommodated in acyl region of the PITP PtdIns and PtdCho was used in the can be the electron density for the for the There are or polar amino acid residues in the region that be to the the structure of the model polar head group of for which are To transfer a PITP to the exchange its bound lipid for a membrane and from the have that are conformational in the C-terminal residues when PITP to lipid membranes (21Tremblay J.M. Helmkamp Jr., G.M. Yarbrough L.R. J. Biol. Chem. 1996; 271: 21075-21080Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). cytosolic PITP is highly to protein bound to membranes is at and In the crystal structure these two sites are of G in the C-terminal have that sites that are to by a structure with a similar to that of J.M. Prot. Sci. 1994; PubMed Scopus Google Scholar). to by a by with a membrane surface. The of of is by the calculated for residues also to be for a PITPs truncated at residues or have more than defined by of by of and binding P.A. Tremblay J.M. Yarbrough L.R. Helmkamp Jr., G.M. 1996; PubMed Scopus Google Scholar). of the structure that be by and of residues a conformational change in the of the have that of one or a amino from the or of a protein have a on protein in R.J. R. M. J.M. G. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, G. J. J. Mol. Biol. 1995; 254: PubMed Scopus Google Scholar). data on the and biological of truncated PITPs are with a of the in the of the and and of the The of PITP to phospholipid substrates is for phospholipid of to 20 C-terminal residues phospholipid these truncated proteins in E. or phosphatidylethanolamine with a P.A. Tremblay J.M. Yarbrough L.R. Helmkamp Jr., G.M. 1996; PubMed Scopus Google Scholar, S. P. D. Cockcroft S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). residues are for lipid they are in for PtdIns and for transfer of is from the PITP structure phospholipids as and phosphatidic acid are bound We that residues be with on transfer that the of the membranes employed in the J.M. P.A. Helmkamp Jr., G.M. Yarbrough L.R. Biochim. Biophys. 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A. 1995; PubMed Scopus Google Scholar). of amino and in observed to PtdIns PtdCho transfer was the of all are conserved among PITPs and data a of the group in a with the and place in the of the fatty a with the conserved in a region to the and polar head group is the of the of the cavity that the acyl of these residues and on PtdIns binding and transfer to and two conserved expression of mammalian PITP in a Jr., A. Bankaitis Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). on these residues the between the and the regulatory participates in with the conserved and with the conserved of the features of as and region constitute the surface. We a model in which the C-terminal region is a of surface. In the between protein and all of the C-terminal region be from A in the and to a of G and to We that the and of the C-terminal region with the protein a for of PITP from the membrane surface. The by which PITP its bound for in the membrane and from the membrane to be A model is one in which the C-terminal region of the protein to the membrane and the a its bound lipid to the membrane surface. The protein-bound lipid is the membrane and replaced by a membrane the C-terminal region and with the of the the and the protein from the membrane surface. The for the membrane and the for a phospholipid be and transfer would be and the of membrane at in PITP of lipid exchange with membranes containing To for other biological of suggest that the protein-bound phospholipid to of a lipid with a on on the regulatory of the residues acid are or conserved among all PITP and proteins interactions are to the of PITP at the head group region of the to the presentation of PtdIns to the of a PtdIns 4-kinase or the of the of a PtdIns to the of a soluble PtdIns 3-kinase or 4-kinase T. Biochim. Biophys. Acta. 1998; PubMed Scopus Google Scholar, L. Biochim. Biophys. Acta. 1998; PubMed Scopus Google Scholar). a of PITP in substrate presentation to a PtdIns 3-kinase has been (10Panaretou C. Domin J. Cockcroft S. Waterman M.D. J. Biol. Chem. 1997; 272: 2477-2485Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). of the or the of the for PITP would that would with the or to the lipid surface. with is the of PITP to and transfer PtdIns P. Insall R. Wilkins A. Cockcroft S. Biochem. J. 2000; 347: 837-843Crossref PubMed Google Helmkamp Jr., G.M. Res. 268: PubMed Scopus Google Scholar). the DALI (30Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3561) Google Scholar) to for proteins structurally similar to the L. Biochem. Sci. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) was identified as a protein with human has as a for structure and of the Y. Biol. 2000; 7: PubMed Scopus Google Scholar). The contains a that the of the protein with on both A β-sheet and two α-helices an that is to be to a single molecule of was included in the crystallization the protein proteins are to and with also common to acute regulatory is to to the membrane and contains a its C-terminus. the sequence of PtdCho transfer protein cytosolic lipid transfer a single In to has a for PtdCho and highly tissue expression in K.W.A. Biochem. J. 1997; 324: 353-360Crossref PubMed Scopus (174) Google Scholar). identified by the DALI the structure of T. T. U. H. K. J. Mol. 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PITP and are similar in exhibit similar in and transport and and highly the two proteins have structural In to the PITPα·PtdCho structure has no bound phospholipid of the PITPα·PtdCho structure a complex lipid enclosed a The of the protein a framework for understanding the of interactions on several its PITP has been to phospholipids from the aqueous environment. ligand by the is by noncovalent The C-terminal region of PITP is to the to and from lipid its regulatory PITP is to lipid substrates to kinases for and structural of PITP and other members of the family are to these and other of PITP interactions with membranes and kinases are to a more understanding of the of PITP in cellular phospholipid

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