Model of Biologically Active Apolipoprotein E Bound to Dipalmitoylphosphatidylcholine

Apolipoprotein (apo)E plays a critical role in cholesterol transport, through high affinity binding to the low density lipoprotein receptor. This interaction requires apoE to be associated with a lipoprotein particle. To determine the structure of biologically active apoE on a lipoprotein particle, we crystallized dipalmitoylphosphatidylcholine particles containing two apoE molecules and determined the molecular envelope of apoE at 10 Å resolution. On the basis of the molecular envelope and supporting biochemical evidence, we propose a model in which each apoE molecule is folded into a helical hairpin with the binding region for the low density lipoprotein receptor at its apex. Apolipoprotein (apo)E plays a critical role in cholesterol transport, through high affinity binding to the low density lipoprotein receptor. This interaction requires apoE to be associated with a lipoprotein particle. To determine the structure of biologically active apoE on a lipoprotein particle, we crystallized dipalmitoylphosphatidylcholine particles containing two apoE molecules and determined the molecular envelope of apoE at 10 Å resolution. On the basis of the molecular envelope and supporting biochemical evidence, we propose a model in which each apoE molecule is folded into a helical hairpin with the binding region for the low density lipoprotein receptor at its apex. Lipoproteins, the major transport vehicles for distribution of triglyceride and cholesterol throughout the body, are divided into different classes based on their apolipoprotein and lipid contents. Although lipoprotein particles have been extensively studied by electron microscopy, neutron diffraction, and x-ray diffraction (for review, see Ref. 1Segrest J.P. Jones M.K. De Loof H. Dashti N. J. Lipid Res. 2001; 42: 1346-1367Abstract Full Text Full Text PDF PubMed Google Scholar), the resolution has not been sufficient to visualize the molecular envelope of individual proteins on the surface of the particle. Lipoproteins containing apolipoprotein (apo) 2The abbreviations used are: apoapolipoproteinLDLlow density lipoprotein(s)LDLRLDL receptorDPPCdipalmitoylphosphatidylcholineDMPCdimyristoylephosphosphatidylcholine. E serve critical functions in plasma cholesterol and triglyceride metabolism and in the transport and redistribution of lipids among various cells, tissues, and organs (2Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3395) Google Scholar). ApoE also plays a major role in neurobiology (3Weisgraber K.H. Mahley R.W. FASEB J. 1996; 10: 1485-1494Crossref PubMed Scopus (277) Google Scholar). Key to many of these functions is the high affinity binding of apoE-containing lipoproteins to the low density lipoprotein (LDL) receptor (LDLR) and other members of the LDLR family. The three common isoforms (apoE2, apoE3, and apoE4) exhibit isoform-specific effects in both cardiovascular and neurodegenerative disease. For example, apoE4 is a major susceptibility factor for Alzheimer disease (4Saunders A.M. Strittmatter W.J. Schmechel D. St. George-Hyslop P.H. Pericak-Vance M.A. Joo S.H. Rosi B.L. Gusella J.F. Crapper-MacLachlan D.R. Alberts M.J. Hulette C. Crain B. Goldgaber D. Roses A.D. Neurology. 1993; 43: 1467-1472Crossref PubMed Google Scholar, 5Corder E.H. Saunders A.M. Strittmatter W.J. Schmechel D.E. Gaskell P.C. Small G.W. Roses A.D. Haines J.L. Pericak-Vance M.A. Science. 1993; 261: 921-923Crossref PubMed Scopus (7342) Google Scholar, 6Strittmatter W.J. Roses A.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4725-4727Crossref PubMed Scopus (447) Google Scholar) and other forms of neurodegeneration (7Mayeux R. Ottman R. Maestre G. Ngai C. Tang M.-X. Ginsberg H. Chun M. Tycko B. Shelanski M. Neurology. 1995; 45: 555-557Crossref PubMed Scopus (522) Google Scholar, 8Slooter A.J.C Tang M.-X. van Duijn C.M. Stern Y. Ott A. Bell K. Breteler M.M.B Van Broeckhoven C. Tatemichi T.K. Tycko B. Hofman A. Mayeux R. J. Am. Med. Assoc. 1997; 277: 818-821Crossref PubMed Google Scholar, 9Fazekas F. Strasser-Fuchs S. Schmidt H. Enzinger C. Ropele S. Lechner A. Flooh E. Schmidt R. Hartung H.-P. J. Neurol. Neurosurg. Psychiatry. 2000; 69: 25-28Crossref PubMed Scopus (82) Google Scholar, 10Drory V.E. Birnbaum M. Korczyn A.D. Chapman J. J. Neurol. Sci. 2001; 190: 17-20Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) and for cardiovascular disease (11Davignon J. Gregg R.E. Sing C.F. Arteriosclerosis. 1988; 8: 1-21Crossref PubMed Google Scholar, 12Eichner J.E. Dunn S.T. Perveen G. Thompson D.M. Stewart K.E. Stroehla B.C. Am. J. Epidemiol. 2002; 155: 487-495Crossref PubMed Scopus (630) Google Scholar). apolipoprotein low density lipoprotein(s) LDL receptor dipalmitoylphosphatidylcholine dimyristoylephosphosphatidylcholine. In the lipid-free state, human apoE (299 residues) has two independently folded structural domains, a 22-kDa N-terminal domain (residues 1-191) containing the LDLR-binding region (residues 140-160 and 172) and a 10-kDa C-terminal domain (residues 216-299) containing the major lipoprotein-binding elements (13Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar, 14Weisgraber K.H. J. Lipid Res. 1990; 31: 1503-1511Abstract Full Text PDF PubMed Google Scholar). The structure of the C-terminal domain is unknown but predicted to be α-helical (15Nolte R.T. Atkinson D. Biophys. J. 1992; 63: 1221-1239Abstract Full Text PDF PubMed Scopus (154) Google Scholar, 16Segrest J.P. Jones M.K. De Loof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. J. Lipid Res. 1992; 33: 141-166Abstract Full Text PDF PubMed Google Scholar, 17Aggerbeck L.P. Wetterau J.R. Weisgraber K.H. Wu C.-S. C. Lindgren F.T. J. Biol. Chem. 1988; 263: 6249-6258Abstract Full Text PDF PubMed Google Scholar), and the structure of the lipid-free N-terminal domain is a four-helix bundle containing antiparallel α-helices (18Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (601) Google Scholar). The residues implicated in LDLR binding are surface-accessible in the crystal structure; however, until it is associated with lipid, apoE does not display high affinity binding to the LDLR (19Innerarity T.L. Pitas R.E. Mahley R.W. J. Biol. Chem. 1979; 254: 4186-4190Abstract Full Text PDF PubMed Google Scholar). Because the N-terminal four-helix bundle opens and then the helices reorganize during lipid binding (20Lu B. Morrow J.A. Weisgraber K.H. J. Biol. Chem. 2000; 275: 20775-20781Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), the final conformation of receptor-active apoE in the lipid-bound state is likely to be dramatically different from that of lipid-free apoE. To determine how binding to a lipoprotein particle activates the LDLR binding site of apoE, we crystallized apoE4 bound to dipalmitoylphosphatidylcholine (DPPC). Here we present an x-ray model of lipid-bound apoE4 at 10 Å resolution. Production and Crystallization of ApoE4·DPPC Particles—Recombinant apoE4 was expressed and purified as described (21Morrow J.A. Arnold K.S. Weisgraber K.H. Protein Expr. Purif. 1999; 16: 224-230Crossref PubMed Scopus (91) Google Scholar). The apoE4·DPPC particles were produced by modification of the method of Jonas (22Matz C.E. Jonas A. J. Biol. Chem. 1982; 257: 4535-4540Abstract Full Text PDF PubMed Google Scholar) (as described in Ref. 23Newhouse Y. Peters-Libeu C. Weisgraber K.H. Acta Crystallogr. F. 2005; 61: 981-984Crossref Scopus (12) Google Scholar). Crystals were obtained with the hanging drop method from a mixture of 24% polyethylene glycol 1000, 20 mm sodium acetate, pH 5.8, and apoE4·DPPC (∼2 mg/ml) containing 1% heptanetriol. Data Collection and Processing—For cryoprotection, apoE4·DPPC crystals were treated with 28% polyethylene glycol 1000, 20 mm sodium acetate, pH 5.8, 1% heptanetriol, and 15% ethylene glycol. Data were collected at beam lines 8.2.1, 8.2.2, and 8.3.1 at the Advanced Light Source with 1.072 Å x-rays. Data were processed with XDS (24Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3243) Google Scholar), and the relevant statistics are summarized in Table 1 and in Ref. 23Newhouse Y. Peters-Libeu C. Weisgraber K.H. Acta Crystallogr. F. 2005; 61: 981-984Crossref Scopus (12) Google Scholar. The space group of the crystals is P21 with cell constants of a = 99 Å, b = 111 Å, c = 79 Å, and β = 113°.TABLE 1Data statistics for apoE·DPPCAll reflectionsaThe total number of reflections that were observed was 3,063 with 881 unique reflections.Strong reflections (I/σ(I) > 3.0)bThe total number of strong reflections was 2,449 with 745 unique reflections.50−10 Å12.5−10 Å50−10 Å12.5−10 ÅCompleteness99%100%83%71%RmergecRmerge = ∑ij (〈Ii 〉 - Iij)2/∑i 〈Ii 〉, where 〈Ii 〉 is the average intensity of the jth reflection and Iij is the intensity of the jth observation of the ith reflection.6.2%21%4.0%11.8%Redundancy3.53.43.53.5<I/(σ(I)>dI is the intensity of the reflection and σ(I) is its estimated error.226.3185.3a The total number of reflections that were observed was 3,063 with 881 unique reflections.b The total number of strong reflections was 2,449 with 745 unique reflections.c Rmerge = ∑ij (〈Ii 〉 - Iij)2/∑i 〈Ii 〉, where 〈Ii 〉 is the average intensity of the jth reflection and Iij is the intensity of the jth observation of the ith reflection.d I is the intensity of the reflection and σ(I) is its estimated error. Open table in a new tab Calculation of Radial Intensity—For each crystal, a series of 0.25° oscillation photos was collected every 10° with an ADSC Q315 detector. Sample images corrected for background scattering from the cryoprotectant are included in the supplemental information. After data collection, the crystal was removed from its fiber loop. The loop was filled with cryoprotectant, and a second set of images was collected for background subtraction. The average radial intensity for each image was calculated with FIT2D v6.0 3Dr. Andy Hammerstey, personal communication. . For the low-resolution calculation, a mask for each image was constructed to eliminate pixels corresponding to the Bragg peaks. For the high-resolution calculation, all pixels below the average diffuse scattering intensity outside the arcs were eliminated to better estimate the average intensity of the arcs. To correct for absorption effects, each radial intensity curve was scaled to the radial intensity curve at 0° using a single scale factor determined by Because the images from the loop the final radial intensity curve for that loop was calculated as the average of the individual radial intensity at each This curve was also scaled to the radial intensity curve from the crystal at The is the the scaled radial intensity curve for each image and the final average radial intensity curve for the loop filled with by molecular were by on of a that the molecular envelope were into the model and into A. A. Acta Crystallogr. D. 2000; PubMed Scopus Google Scholar) as a series of The molecular was of the and In to of the molecular surface were the and of the the of and the The of the of the and be for a and for a The is the of the of an has an of The was 20 and The and the were and The of was and and the was and The was in of Å, the of was in of and the was in of a of their of of Å, and were used for the of and The and the were in of Calculation of the the diffuse scattering observed from the model particles were constructed by with the of the particle. For example, a particle was as two of filled with that the have a in to the of the Although model is and does not a of the diffuse scattering from a particle, it is sufficient to determine the of the diffuse scattering associated with the and the the The diffuse scattering from the of the particle was by in at a of Å from the of the particle. were the model was to the of the molecular and then the molecule was with the P21 The of each cell was calculated with the from and a For each calculation, the of was and then with LDLR-binding of ApoE4·DPPC was for structural it has a high and ApoE4·DPPC particles two molecules of apoE4 and molecules of Y. Peters-Libeu C. Weisgraber K.H. Acta Crystallogr. F. 2005; 61: 981-984Crossref Scopus (12) Google Scholar). The binding of these particles to the LDLR was determined in a with LDL and a model (19Innerarity T.L. Pitas R.E. Mahley R.W. J. Biol. Chem. 1979; 254: 4186-4190Abstract Full Text PDF PubMed Google Scholar). particles are biologically binding with high affinity to the LDLR Y. Peters-Libeu C. Weisgraber K.H. Acta Crystallogr. F. 2005; 61: 981-984Crossref Scopus (12) Google Scholar). a diffraction from the apoE4·DPPC in two of Bragg and diffuse scattering of and arcs diffuse scattering are to the scattering observed from of In various the scattering from an and Å PubMed Scopus Google Scholar) and and Å D.M. The of to Scholar). in the as the crystals a at Å and two at and Å not that strong diffuse scattering from the apoE4·DPPC crystals is of the molecules the apoE4·DPPC In the Å was as a of diffraction two of in PubMed Scopus Google Scholar). the of diffuse scattering at and Å were associated with in the of the D.M. The of to Scholar). In the apoE4·DPPC a of high intensity scattering was observed at Å, but the diffuse scattering on Å was to arcs a The intensity of these was of the crystal with to the x-ray the intensity of the from the average intensity This is the of diffraction, two Bragg the in the intensity of the Å arcs is of crystal This of diffuse scattering is with the of in which the molecules are in a M. B. Weisgraber K. M. 1995; PubMed Scopus Google Scholar, E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the particle and from the molecular the predicted diffuse scattering of a Å high and Å in with to the was The strong on the in the and the in the at and Å The of the two is to distribution of in the model and The scattering from the of the is to the and as the where is an the and the are in their that the predicted of intensity for a the crystal is it is that the of diffuse scattering from the apoE4·DPPC crystals does not from lipid in a The diffuse scattering be by using and a model for the Å in filled with Å with a of at where is an Because an of the diffuse scattering of molecules that from the average Å the that the particle is the of the particles from the diffuse scattering likely from to scattering to the in a model is to the of the that the Å in the diffraction from a of a of the the particle. Although is to model the of the for a of a of the is that the particle is into an in a that the of the an the diffuse scattering observed from the apoE4·DPPC crystals is with the diffuse scattering from a particle a particle containing a This is not of the crystal, that that the apoE4·DPPC particles are which is with the of Bragg the crystals into particles in to used to the crystals Y. Peters-Libeu C. Weisgraber K.H. Acta Crystallogr. F. 2005; 61: 981-984Crossref Scopus (12) Google Scholar), the diffuse scattering from these crystals does not from of The from the model are with the of the are on the surface of a Å in an average of Å for the a the of estimated of molecules in at low the particle is as a the molecular of the is For the molecular of the of is estimated to be at D.M. The of to Scholar), for the crystals are to K. Because of the of the the estimated surface is the corresponding of observed for at K. molecular and an surface group were observed in of in which space the is filled with molecules J.F. S. Biophys. 2000; PubMed Scopus Google Scholar). likely in apoE4·DPPC with the surface the to the the folded a The of the apoE4·DPPC particles was electron of particles with of Å Y. Peters-Libeu C. Weisgraber K.H. Acta Crystallogr. F. 2005; 61: 981-984Crossref Scopus (12) Google Scholar), with the crystals are by the state for the in Ref. 23Newhouse Y. Peters-Libeu C. Weisgraber K.H. Acta Crystallogr. F. 2005; 61: 981-984Crossref Scopus (12) Google Scholar, different of and lipid to during in a of apoE4·DPPC particles with different likely from in and of the diffuse scattering that the and the lipid to to we used for the of the lipid the of diffuse scattering that the does not a as predicted by of the is the cell and to the Bragg with not the that other apoE-containing particles as with apoE the of the ApoE a of these particles for was the of the lipid that the Bragg from diffraction by the This a unique to the structure of the molecular envelope of apoE by molecular of apoE bound to particles have been the and M. B. Weisgraber K. M. 1995; PubMed Scopus Google Scholar, E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the apoE is folded into a series of helices the of a with the helices to the In the apoE is folded into a single that the with the to the of the 10 Å the model a of Å and the model a of and Because the two in the apoE4·DPPC crystals supplemental it is that apoE forms a that the particle. of a were used in a molecular model was by at a density an envelope by by the and For each the molecular was The and of each on the and of the For each of the and the were 1 Å for 1 Å for and Å for of a for the that the model has a be obtained for different of the and with high to a that the with the of the is to a of 1 Å, and the of to The for of were and the were for all reflections and 10 it that 10 Å data the of the envelope with of the in the in the of The molecular envelope was an that a with a in the The a second envelope that the in an that that the surface of the particle is with the diffuse scattering In the apoE molecules the surface of the with an the of which with the of the particles estimated by electron Y. Peters-Libeu C. Weisgraber K.H. Acta Crystallogr. F. 2005; 61: 981-984Crossref Scopus (12) Google Scholar). The the crystal but the are to Å, which is the at resolution. Although 10 Å resolution is to the structure the the and the of the electron density from a of helices at 10 Å as the helices in the crystal structure M. 2002; PubMed Scopus Google Scholar). This each apoE molecule be as a of helices folded into a and on of the estimated that the helical of apoE is in particles but in the lipid-free the molecular envelope likely a series of helices and with of the the from diffraction and the of the envelope from molecular that a of the of the apoE molecule the surface of the Because the of the particle not until Å below the on the J.F. S. Biophys. 2000; PubMed Scopus Google Scholar), the estimate of the of the the of the of the particle the of the surface residues of apoE the the the molecules the the To be folded into the structure of apoE likely the structure of J.A. Brouillette C.G. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar) of in the of a M. 2002; PubMed Scopus Google Scholar). In both of these the proteins are of helices with a This is in to the of how apoE to In other the of the residues of apoE with the M. B. Weisgraber K. M. 1995; PubMed Scopus Google Scholar, E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the supporting these used an N-terminal of apoE and the different and of the C-terminal domain on the apoE4·DPPC particles for the of to apoE the is to the molecule in to a helical hairpin to This conformation the of the molecular as each of the the residues in apoE. The radial of the molecular envelope is with two helices of which the of the N-terminal four-helix bundle (residues on these a helical hairpin model of apoE be that is with the helical by the observed structure of the N-terminal and the predicted structure of the C-terminal domain (15Nolte R.T. Atkinson D. Biophys. J. 1992; 63: 1221-1239Abstract Full Text PDF PubMed Scopus (154) Google Scholar, 16Segrest J.P. Jones M.K. De Loof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. J. Lipid Res. 1992; 33: 141-166Abstract Full Text PDF PubMed Google Scholar, 17Aggerbeck L.P. Wetterau J.R. Weisgraber K.H. Wu C.-S. C. Lindgren F.T. J. Biol. Chem. 1988; 263: 6249-6258Abstract Full Text PDF PubMed Google Scholar). the is to the of the molecular envelope and The C-terminal domain to the surface the N-terminal domain to the of the residues in the LDL This is with two that the with the site at and the C-terminal binding site K.H. Mahley R.W. R.W. J. Biol. Chem. 261: Full Text PDF PubMed Google Scholar) and that the C-terminal domain the binding of apoE to and has a for lipid the N-terminal domain F. Anantharamaiah G.M. Weisgraber K.H. S. J. Lipid Res. 2002; 43: Full Text Full Text PDF PubMed Scopus Google Scholar). a helical hairpin model for molecular the as the molecular envelope with a of and a of The of a of the to and the to in the of with a of the to and the to The molecular envelope at 10 Å resolution does not by determine the helical hairpin structure from a number of different of helices and each of the the of the helical the be of the and C-terminal be in it is to the that structural the molecule in resolution structure be to The LDLR-binding at the of the the of the helical hairpin the of the N-terminal bundle all the elements of the LDLR binding site into (13Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar, J.A. Arnold K.S. J. T.L. Weisgraber K.H. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). In lipid-free apoE, the residues at the of the of the N-terminal domain (residues and in the loop the two are by residues were as the the of the of the C-terminal was to the of the of the N-terminal In the was to the other residues on and the helical hairpin model lipid is for high affinity binding of apoE to the The of residues to the loop is by of the and structure of of apoE. of apoE at receptor binding but apoE at is receptor-active A. Mahley R.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In the the structure of residues of the helical of these residues is an on the the and these both lipid-free and apoE R. R. Weisgraber K. R. D. E. R. J. Lipid Res. 1995; Full Text PDF PubMed Google Scholar). of residues bound to that of with a at C.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and through is also the of a in in the lipid-free crystal of the N-terminal domain M. M. S. Weisgraber K.H. B. Protein Sci. 2000; PubMed Scopus Google Scholar). The and of in different crystal that the conformation of residues C-terminal to on their The of residues also that likely a are a unique the apoE In the lipid-free residues are with the residues in of residues is The with the and structure of various apoE is with a model in which residues the the two The hairpin model a role for of In the and are critical in the Although are many to the at of the residues a to the the envelope and into with the of the residues implicated in LDLR binding the LDLR a conformation of loop. the hairpin structure by binding to apoE be lipid-bound to with high affinity to the LDLR the of the molecular The molecular of region resolution In of the diffraction of apoE4·DPPC crystals that the particles are with a of Å the of the The molecular envelope of apoE is an the and C-terminal domains, are and that of the residues in the helices an the two helices with the of the particle. of apoE as a helical hairpin the molecular envelope and a distribution of and residues that apoE with the and the and the of and structural that the LDLR binding site is to of the at the of the helical hairpin and that the of hairpin loop from lipid is critical for high affinity LDLR binding This not have been of through the of the Advanced Light and the and for data and for and for and and for with