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• W2125103697 abstract "Human apolipoprotein CIII (apoCIII) is a surface component of chylomicrons, very low density lipoproteins, and high density lipoproteins. ApoCIII inhibits lipoprotein lipase as well as binding of lipoproteins to cell surface heparan sulfate proteoglycans and receptors. High levels of apoCIII are often correlated with elevated levels of blood lipids (hypertriglyceridemia). Here, we report the three-dimensional NMR structure and dynamics of human apo-CIII in complex with SDS micelles, mimicking its natural lipid-bound state. Thanks to residual dipolar coupling data, the first detailed view is obtained of the structure and dynamics of an intact apolipoprotein in its lipid-bound state. ApoCIII wraps around the micelle surface as a necklace of six ∼10-residue amphipathic helices, which are curved and connected via semiflexible hinges. Three positively charged (Lys) residues line the polar faces of helices 1 and 2. Interestingly, their three-dimensional conformation is similar to that of the low density lipoprotein receptor binding motifs of apoE/B and the receptor-associated protein. At the C-terminal side of apoCIII, an array of negatively charged residues lines the polar faces of helices 4 and 5 and the adjacent flexible loop. Sequence comparison shows that this asymmetric charge distribution along the solvent-exposed face of apoCIII as well as other structural features are conserved among mammals. This structure provides a template for exploration of molecular mechanisms by which human apoCIII inhibits lipoprotein lipase and receptor binding. Human apolipoprotein CIII (apoCIII) is a surface component of chylomicrons, very low density lipoproteins, and high density lipoproteins. ApoCIII inhibits lipoprotein lipase as well as binding of lipoproteins to cell surface heparan sulfate proteoglycans and receptors. High levels of apoCIII are often correlated with elevated levels of blood lipids (hypertriglyceridemia). Here, we report the three-dimensional NMR structure and dynamics of human apo-CIII in complex with SDS micelles, mimicking its natural lipid-bound state. Thanks to residual dipolar coupling data, the first detailed view is obtained of the structure and dynamics of an intact apolipoprotein in its lipid-bound state. ApoCIII wraps around the micelle surface as a necklace of six ∼10-residue amphipathic helices, which are curved and connected via semiflexible hinges. Three positively charged (Lys) residues line the polar faces of helices 1 and 2. Interestingly, their three-dimensional conformation is similar to that of the low density lipoprotein receptor binding motifs of apoE/B and the receptor-associated protein. At the C-terminal side of apoCIII, an array of negatively charged residues lines the polar faces of helices 4 and 5 and the adjacent flexible loop. Sequence comparison shows that this asymmetric charge distribution along the solvent-exposed face of apoCIII as well as other structural features are conserved among mammals. This structure provides a template for exploration of molecular mechanisms by which human apoCIII inhibits lipoprotein lipase and receptor binding. Apolipoproteins consist of five major classes, apo-A 2The abbreviations used are: apo, apolipoprotein; LPL, lipoprotein lipase; LDL, low density lipoprotein; HDL, high density lipoprotein; RDC, residual dipolar coupling; NOE, nuclear Overhauser effect; r.m.s., root mean square; RAP, receptor-associated protein. through -E, and several subclasses. They are designed for lipid transport in blood and are attached to lipid droplets (lipoproteins) through amphipathic helices that intercalate into the single layer of phospholipids and cholesterol covering the droplet (1Segrest J.P. Jones M.K. Deloof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. J. Lipid Res. 1992; 33: 141-166Abstract Full Text PDF PubMed Google Scholar, 2Narayanaswami V. Ryan R.O. Biochim. Biophys. Acta. 2000; 1483: 15-36Crossref PubMed Scopus (159) Google Scholar, 3Saito H. Lund-Katz S. Phillips M.C. Prog. Lipid Res. 2004; 43: 350-380Crossref PubMed Scopus (188) Google Scholar). The apolipoproteins regulate blood lipid levels by interaction with specialized endocytotic receptors and by modulating enzyme activities and lipid exchange reactions (4Jong M.C. Hofker M.H. Havekes L.M. Arterioscl. Thromb. Vasc. Biol. 1999; 19: 472-484Crossref PubMed Scopus (437) Google Scholar). They are therefore in focus with regard to disturbances in blood lipid metabolism (dyslipidemias), which in turn are associated with metabolic disorders like obesity, insulin resistance, diabetes type 2, and cardiovascular disease. Apolipoprotein CIII (apoCIII) is the most abundant C-apolipoprotein in humans (2Narayanaswami V. Ryan R.O. Biochim. Biophys. Acta. 2000; 1483: 15-36Crossref PubMed Scopus (159) Google Scholar, 3Saito H. Lund-Katz S. Phillips M.C. Prog. Lipid Res. 2004; 43: 350-380Crossref PubMed Scopus (188) Google Scholar, 4Jong M.C. Hofker M.H. Havekes L.M. Arterioscl. Thromb. Vasc. Biol. 1999; 19: 472-484Crossref PubMed Scopus (437) Google Scholar). It is found on very low density lipoproteins, chylomicrons, and high density lipoproteins (HDL). ApoCIII is predominantly expressed in liver and intestine from a gene cluster important for lipid regulation (ApoAI, -AIV, -AV, and -CIII) (4Jong M.C. Hofker M.H. Havekes L.M. Arterioscl. Thromb. Vasc. Biol. 1999; 19: 472-484Crossref PubMed Scopus (437) Google Scholar, 5Talmud P.J. Hawe E. Martin S. Olivier M. Miller G.J. Rubin E.M. Pennacchio L.A. Humphries S.E. Hum. Mol. Genet. 2002; 11: 3039-3046Crossref PubMed Scopus (343) Google Scholar). ApoCIII inhibits lipoprotein lipase (LPL) and receptor-mediated endocytosis of lipoprotein particles by competing for space at the surface of the lipoproteins and by interfering with their binding to endothelial proteoglycans and to specific lipoprotein receptors (2Narayanaswami V. Ryan R.O. Biochim. Biophys. Acta. 2000; 1483: 15-36Crossref PubMed Scopus (159) Google Scholar, 4Jong M.C. Hofker M.H. Havekes L.M. Arterioscl. Thromb. Vasc. Biol. 1999; 19: 472-484Crossref PubMed Scopus (437) Google Scholar, 6Clavey V. Lestavel-Delattre S. Copin C. Bard J.M. Fruchart J.C. Arterioscl. Thromb. Vasc. Biol. 1995; 15: 963-971Crossref PubMed Scopus (167) Google Scholar). Overexpression of apoCIII in transgenic mice leads to severely increased plasma triglyceride levels due to accumulation of very low density lipoprotein-like lipoprotein remnants with increased apo-CIII and decreased apoE content compared with controls (7Ito Y. Azrolan N. Oconnell A. Walsh A. Breslow J.L. Science. 1990; 249: 790-793Crossref PubMed Scopus (453) Google Scholar). Overexpression of apoCIII in apoE-/- mice leads to similar results, demonstrating that the effects are not only connected to displacement of apoE (8Ebara T. Ramakrishnan R. Steiner G. Shachter N.S. J. Clin. Invest. 1997; 99: 2672-2681Crossref PubMed Scopus (165) Google Scholar). Knock-out of the apoCIII gene in mice leads to reduced levels of lipoproteins in blood (9Maeda N. Li H. Lee D. Oliver P. Quarfordt S.H. Osada J. J. Biol. Chem. 1994; 269: 23610-23616Abstract Full Text PDF PubMed Google Scholar). In human subjects, increased levels of apoCIII are often correlated to increased levels of triglycerides in blood and in turn to insulin resistance, cardiovascular disease, and diabetes type 2 (4Jong M.C. Hofker M.H. Havekes L.M. Arterioscl. Thromb. Vasc. Biol. 1999; 19: 472-484Crossref PubMed Scopus (437) Google Scholar). Recent data suggest that apoCIII and apoAV are important opposing modulators of plasma triglyceride metabolism (5Talmud P.J. Hawe E. Martin S. Olivier M. Miller G.J. Rubin E.M. Pennacchio L.A. Humphries S.E. Hum. Mol. Genet. 2002; 11: 3039-3046Crossref PubMed Scopus (343) Google Scholar, 10van Dijk K.W. Rensen P.C.N. Voshol P.J. Havekes L.M. Curr. Opin. Lipodol. 2004; 15: 239-247Crossref PubMed Scopus (127) Google Scholar). ApoCIII was found to directly affect insulin secretion by interfering with voltage-gated calcium channels in pancreatic β-cells (11Juntti-Berggren L. Refai E. Appelskog I. Andersson M. Imreh G. Dekki N. Uhles S. Yu L. Griffiths W.J. Zaitsev S. Leibiger I. Yang S.N. Olivecrona G. Jornvall H. Berggren P.O. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10090-10094Crossref PubMed Scopus (76) Google Scholar). Interestingly, mutations in the apoCIII promoter are connected with longevity (12Kaiser J. Science. 2004; 306: 1284Google Scholar). Lipid interaction is required for solubility as well as correct folding and function of most apolipoproteins (1Segrest J.P. Jones M.K. Deloof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. J. Lipid Res. 1992; 33: 141-166Abstract Full Text PDF PubMed Google Scholar, 2Narayanaswami V. Ryan R.O. Biochim. Biophys. Acta. 2000; 1483: 15-36Crossref PubMed Scopus (159) Google Scholar, 3Saito H. Lund-Katz S. Phillips M.C. Prog. Lipid Res. 2004; 43: 350-380Crossref PubMed Scopus (188) Google Scholar). In addition, cumulative evidence suggests that apolipoproteins are dynamic and span multiple conformations required for their functions (13Gursky O. Curr. Opin. Lipodol. 2005; 16: 287-294Crossref PubMed Scopus (40) Google Scholar). Structural studies of apolipoproteins have therefore been as challenging as studies of membrane proteins, and high resolution structure information has remained relatively scarce. To date, apolipoprotein structures are available of the 22-kDa N-terminal domain of human/mouse apoE (14Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (601) Google Scholar, 15Segelke W. Forstner M. Knapp M. Trakhanov S. Parkin S. Newhouse Y. Bellamy H.D. Weisgraber K.H. Rupp B. Protein Sci. 2000; 9: 886-897Crossref PubMed Scopus (48) Google Scholar, 16Hatters D.M. Peters-Libeu C.A. Weisgraber K.H. J. Biol. Chem. 2005; 280: 26477-26482Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), Δ(1–43)apoAI (17Borhani D.W. Rogers D.P. Engler J.A. Brouillette C.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12291-12296Crossref PubMed Scopus (414) Google Scholar) and of three intact apolipoproteins, apoAI (18Ajees A.A. Anantharamatah G.M. Mishra V.K. Hussain M.M. Murthy H.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2126-2131Crossref PubMed Scopus (194) Google Scholar), insect LpIII (19Wang J. Sykes B.D. Ryan R.O. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1188-1193Crossref PubMed Scopus (103) Google Scholar, 20Fan D. Zheng Y. Yang D. Wang J. J. Biol. Chem. 2003; 278: 21212-21218Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and apoAII (21Kumar M.S. Carson M. Hussain M.M. Murthy H.M. Biochemistry. 2002; 41: 11681-11691Crossref PubMed Scopus (35) Google Scholar), all in the lipid-free state. In the lipid-bound state, only recently the first low resolution (10 Å) x-ray model has been reported of an intact apolipoprotein, apoE (22Peters-Libeu C.A. Newhouse Y. Hatters D.M. Weisgraber K.H. J. Biol. Chem. 2006; 281: 1073-1079Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Also reported is the three-dimensional structure and dynamics of apoCII bound to micelles derived from NMR relaxation data (23Zdunek J. Martinez G.V. Schleucher J. Lycksell P.O. Yin Y. Nilsson S. Shen Y. Olivecrona G. Wijmenga S. Biochemistry. 2003; 42: 1872-1889Crossref PubMed Scopus (48) Google Scholar). In addition, NMR has provided information on helix boundaries and local structure of lipid-bound intact apoCII (24MacRaild C.A. Howlett G.J. Gooley P.R. Biochemistry. 2004; 43: 8084-8093Crossref PubMed Scopus (49) Google Scholar), apoCI (25Rozek A. Sparrow J.T. Weisgraber K.H. Cushley R.J. Biochemistry. 1999; 38: 14475-14484Crossref PubMed Scopus (40) Google Scholar), and apoAI (26Okon M. Frank P.G. Marcel Y.L. Cushley R.J. FEBS Lett. 2002; 517: 139-143Crossref PubMed Scopus (32) Google Scholar). No high resolution structural and dynamics information has yet been derived on intact apoCIII. Human apoCIII consists of 79 amino acid residues. Structure predictions have indicated that two amphipathic helices are likely to be formed (1Segrest J.P. Jones M.K. Deloof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. J. Lipid Res. 1992; 33: 141-166Abstract Full Text PDF PubMed Google Scholar, 27Liu H.Q. Talmud P.J. Lins L. Brasseur R. Olivecrona G. Peelman F. Vandekerckhove J. Rosseneu M. Labeur C. Biochemistry. 2000; 39: 9201-9212Crossref PubMed Scopus (36) Google Scholar). There is no consensus about which regions are most important for attachment to lipids (27Liu H.Q. Talmud P.J. Lins L. Brasseur R. Olivecrona G. Peelman F. Vandekerckhove J. Rosseneu M. Labeur C. Biochemistry. 2000; 39: 9201-9212Crossref PubMed Scopus (36) Google Scholar, 28Sparrow J.T. Pownall H.J. Hsu F.J. Blumenthal L.D. Culwell A.R. Gotto A.M. Biochemistry. 1977; 16: 5427-5431Crossref PubMed Scopus (42) Google Scholar, 29Lins L. Flore C. Chapelle L. Talmud P.J. Thomas A. Brasseur R. Protein Eng. 2002; 15: 513-520Crossref PubMed Scopus (42) Google Scholar). The structurally related apoCII is known to interact with LPL and activate this enzyme via the C-terminal helix (30Shen Y. Lookene A. Nilsson S. Olivecrona G. J. Biol. Chem. 2002; 277: 4334-4342Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), most likely in a dynamic fashion (23Zdunek J. Martinez G.V. Schleucher J. Lycksell P.O. Yin Y. Nilsson S. Shen Y. Olivecrona G. Wijmenga S. Biochemistry. 2003; 42: 1872-1889Crossref PubMed Scopus (48) Google Scholar). ApoCIII inhibits LPL, but direct binding has not been shown (27Liu H.Q. Talmud P.J. Lins L. Brasseur R. Olivecrona G. Peelman F. Vandekerckhove J. Rosseneu M. Labeur C. Biochemistry. 2000; 39: 9201-9212Crossref PubMed Scopus (36) Google Scholar). ApoCIII also inhibits binding of apoE/B-containing lipoproteins to members of the LDL receptor family (2Narayanaswami V. Ryan R.O. Biochim. Biophys. Acta. 2000; 1483: 15-36Crossref PubMed Scopus (159) Google Scholar, 6Clavey V. Lestavel-Delattre S. Copin C. Bard J.M. Fruchart J.C. Arterioscl. Thromb. Vasc. Biol. 1995; 15: 963-971Crossref PubMed Scopus (167) Google Scholar). Finally, apoCIII binds, like apoE, apoD, and apoAI, to the HDL receptor SR-BI (31Zannis V.I. Chroni A. Krieger M. J. Mol. Med. 2006; 84: 276-294Crossref PubMed Scopus (310) Google Scholar, 32Xu S. Laccotripe M. Huang X. Rigotti A. Zannis V.I. Krieger M. J. Lipid Res. 1997; 38: 1289-1298Abstract Full Text PDF PubMed Google Scholar). Here, we present the three-dimensional structure and dynamics of intact human apoCIII in complex with SDS micelles, derived from a combination of NMR data: NOEs, 13C chemical shift-derived backbone dihedral angles, 3JHNHα couplings, NMR multiple-field 15N-spin backbone relaxation, and 15N/1H residual dipolar couplings (RDCs). Thanks to the RDC data, the first detailed view is obtained of the structure and dynamics of an intact apoliprotein in a lipid-bound state. This structure provides a template for exploration of the molecular mechanisms by which human apoCIII inhibits LPL and receptor binding. ApoCIII was expressed from a pET23b vector (a kind gift from Dr. Philippa Talmud (London, UK)), containing the full-length cDNA for human apoCIII, including the sequence for a His6 tag at the 3′-end (C-terminal His6 tag) preceded by two additional residues (Leu and Glu) (27Liu H.Q. Talmud P.J. Lins L. Brasseur R. Olivecrona G. Peelman F. Vandekerckhove J. Rosseneu M. Labeur C. Biochemistry. 2000; 39: 9201-9212Crossref PubMed Scopus (36) Google Scholar). A detailed protocol is presented in the supplemental material. The 13C/15N-labeled M13 coat protein was expressed and purified as described elsewhere (33Papavoine C.H.M. Christiaans B.E.C. Folmer R.H.A. Konings R.N.H. Hilbers C.W. J. Mol. Biol. 1998; 282: 401-419Crossref PubMed Scopus (69) Google Scholar, 34Papavoine C.H.M. Remerowski M.L. Horstink L.M. Konings R.N.H. Hilbers C.W. vandeVen F.J.M. Biochemistry. 1997; 36: 4015-4026Crossref PubMed Scopus (55) Google Scholar). The NMR sample of apoCIII in complex with SDS micelles was prepared from lyophilized 15N/13C-labeled apoCIII as described (23Zdunek J. Martinez G.V. Schleucher J. Lycksell P.O. Yin Y. Nilsson S. Shen Y. Olivecrona G. Wijmenga S. Biochemistry. 2003; 42: 1872-1889Crossref PubMed Scopus (48) Google Scholar), leading to the final ApoCIII-NMR sample: ∼0.5 mm apoCIII, 180 mm SDS-d25, 8% D2O, and 10 mm deuterated sodium acetate buffer (pH 5.0). In this final sample, the apo-CIII/micelle ratio is 1:6, assuming 60 SDS molecules/micelle (23Zdunek J. Martinez G.V. Schleucher J. Lycksell P.O. Yin Y. Nilsson S. Shen Y. Olivecrona G. Wijmenga S. Biochemistry. 2003; 42: 1872-1889Crossref PubMed Scopus (48) Google Scholar). For RDC measurements, we prepared a similar second sample (apoCIII-RDC), except for a slightly higher buffer concentration (50 mm). While keeping the apoCIII/SDS micelle ratio as low as possible (here 1:6) to prevent protein-protein interactions (23Zdunek J. Martinez G.V. Schleucher J. Lycksell P.O. Yin Y. Nilsson S. Shen Y. Olivecrona G. Wijmenga S. Biochemistry. 2003; 42: 1872-1889Crossref PubMed Scopus (48) Google Scholar), relatively low SDS and buffer concentrations were used. This considerably reduced the overall tumbling time of the apoCIII-micelle complex and improved three-dimensional NMR spectra, as will be described elsewhere in more detail. All NMR experiments were carried out at 42.7 °C, determined to be optimal via 15N/1H HSQC experiments. Prior to each experiment, the temperature was calibrated using tetramethylammonium. The NMR sample for the 13C/15N-labeled M13 coat protein (gVIIIp) in complex with SDS micelles (M13-NMR) was similar to that for apoCIII. For RDC measurements, a similar second sample (M13-RDC) was prepared. The NMR experiments for assignment and derivation of experimental restraints are summarized in Table S4. Briefly, for backbone resonance assignments, 600-MHz CBCA(CO)NH, HNCACB, and HNCO were recorded (35Sattler M. Schleucher J. Griesinger C. Prog. Nucl. Magn. Reson. Spectrosc. 1999; 34: 93-158Abstract Full Text Full Text PDF Scopus (1399) Google Scholar) and a 600-MHz three-dimensional HNHA for 3JHαHN couplings (36Vuister G.W. Bax A. J. Am. Chem. Soc. 1993; 115: 7772-7777Crossref Scopus (1055) Google Scholar). To confirm the backbone assignments and derive NOE distance restraints, 800-MHz 1H/15N NOESY-HSQC and 1H/15N HSQC-NOESY-HSQC were recorded (35Sattler M. Schleucher J. Griesinger C. Prog. Nucl. Magn. Reson. Spectrosc. 1999; 34: 93-158Abstract Full Text Full Text PDF Scopus (1399) Google Scholar). All NMR spectra were processed using NMRPipe (37Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11638) Google Scholar) and analyzed using XEASY (38Bartels C. Xia T.H. Billeter M. Guntert P. Wuthrich K. J. Biomol. NMR. 1995; 6: 1-10Crossref PubMed Scopus (1607) Google Scholar) and NMRDraw (37Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11638) Google Scholar). Further details on restraint extraction are described under “Structure Calculations.” RDCs were measured in stretched polyacrylamide gels via a procedure modified from previously published ones (39Chou J.J. Gaemers S. Howder B. Louis J.M. Bax A. J. Biomol. NMR. 2001; 21: 377-382Crossref PubMed Scopus (222) Google Scholar) (i.e. practical procedures were developed to control alignment magnitude and buffer conditions (pH and salt)). Briefly, 4% (w/v) polyacrylamide gel (acrylamide/bisacrylamide, 37.5:1) was polymerized in the presence of apoCIII-SDS micelles (apoCIII-RDC sample) in a cylindrical Teflon tube with a 6-mm internal diameter. To induce alignment, the 6-mm diameter gel was then squeezed into an open-ended NMR tube (internal diameter 4.2 mm) using a laboratory-made funnel device (which will be described elsewhere). The HN-N RDCs were measured from five sets of 1H/15N 800-MHz IPAP-HSQCs (40Ottiger M. Delaglio F. Bax A. J. Magn. Reson. 1998; 131: 373-378Crossref PubMed Scopus (845) Google Scholar). In addition, a decoupled 1H/15N HSQC was recorded, so that RDCs could be calculated as twice the difference of the peak positions of the (sharp) “down-component” in the IPAP and decoupled HSQC. The average r.m.s. deviation of the RDCs was 0.8 Hz. The RDC data on the M13 coat protein were measured in a similar fashion. 15N T1 and T1ρ and 15N(1H) NOE measurements (23Zdunek J. Martinez G.V. Schleucher J. Lycksell P.O. Yin Y. Nilsson S. Shen Y. Olivecrona G. Wijmenga S. Biochemistry. 2003; 42: 1872-1889Crossref PubMed Scopus (48) Google Scholar, 41Farrow N.A. Muhandiram R. Singer A.U. Pascal S.M. Kay C.M. Gish G. Shoelson S.E. Pawson T. Formankay J.D. Kay L.E. Biochemistry. 1994; 33: 5984-6003Crossref PubMed Scopus (2018) Google Scholar) on the apoCIII-NMR sample were carried out at 600 and 800 MHz. Two and three sets of 15N T1 experiments were recorded with relaxation delays of 16, 256, 384, 512, 640, 768, 896, 1024, and 1280 ms at 600 and 800 MHz, respectively. Two sets of 15N T1ρ experiments (spin-lock field 3 kHz) were recorded using delays of 16, 32, 48, 64, 96, and 128 ms at 600 and 800 MHz each. The 15N (1H) NOE was acquired thrice at 600 MHz and twice at 800 MHz. The data points were recorded in an interleaved manner to minimize effects of spectrometer drift and temperature fluctuations. All of the data were processed with NMRPipe (37Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11638) Google Scholar). 15N T1 and T1ρ were calculated by nonlinear fittings of the delay-dependent peak intensities to an exponential decay function. 15NT1ρ values were corrected for off-resonance effects. 15N(1H) NOE values were calculated as the intensity ratio of cross-peaks from pairs of spectra acquired with and without 1H presaturation. The data obtained from the duplicate or triplicate measurements were averaged, and errors were derived. The T1, T1ρ, and NOE data were analyzed via PINATA (23Zdunek J. Martinez G.V. Schleucher J. Lycksell P.O. Yin Y. Nilsson S. Shen Y. Olivecrona G. Wijmenga S. Biochemistry. 2003; 42: 1872-1889Crossref PubMed Scopus (48) Google Scholar, 42Larsson G. Schleucher J. Onions J. Hermann S. Grundstrom T. Wijmenga S.S. Biophys. J. 2005; 89: 1214-1226Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 43Larsson G. Martinez G. Schleucher J. Wijmenga S.S. J. Biomol. NMR. 2003; 27: 291-312Crossref PubMed Scopus (22) Google Scholar). To derive a structure model of apoCIII in complex with SDS micelles, we employed a protocol that is essentially as described previously for the apoCII-SDS micelle complex (23Zdunek J. Martinez G.V. Schleucher J. Lycksell P.O. Yin Y. Nilsson S. Shen Y. Olivecrona G. Wijmenga S. Biochemistry. 2003; 42: 1872-1889Crossref PubMed Scopus (48) Google Scholar). The main difference is that the restraints that define the relative helix orientations are now derived from RDCs, whereas previously they originated from the variation in the helix-specific correlation times as derived from 15N relaxation data. The protocol consists of two main parts. In this section (i), an overview is presented of the protocol, whereas in sections ii and iii, elements of the protocol are described in detail. Part I. Structure Calculations Performed Entirely within X-PLOR-NIH 2.1 with Its Standard Simulated Annealing Protocol in Torsion Angle Space and NMR Restraints Corresponding to NOE, CDIH, JCOUP, and SANI (RDC) Terms in the Available Potential—Starting from an α-helical conformation (section iia) a set of 100 apoCIII structures were calculated using X-PLOR-NIH 2.1 with its standard SA protocol in torsion angle space (section iie) (44Schwieters C.D. Kuszewski J.J. Tjandra N. Clore G.M. J. Magn. Reson. 2003; 160: 65-73Crossref PubMed Scopus (1882) Google Scholar). In these calculations, restraints obtained from the following NMR experiments/data were used: NOEs (section iib), 13C chemical shift-derived (via TALOS) backbone dihedral angles (CDIH) (section iic), 3J. Zdunek, M. Lindgren, M. Karlsson, P. O. Westlund, and S. Wijmenga, manuscript in preparation. JHNHα couplings (JCOUP) (section iic), and 15N/1H RDCs (SANI) (section iid). The resulting set of structures shows good structure characteristics (Table S2a). The structures show well defined secondary structures of the helices (data not shown). However, the relative orientations of the helices are not fully defined by these restraints. This is due in part to the local nature of the “classical” NMR restraints and the lack of long range NOEs (as evident from separate calculations without RDCs). That inclusion of the RDCs neither fully defines the relative helix orientations is due to the intrinsic symmetry relations in the geometric equations of the RDCs. Hence, to fully define the three-dimensional structure of the apoCIII-micelle complex, the micelle has to be taken into account. This is done in Part II. To proceed, the 10 lowest energy structures were submitted to part II. Part II. Structure Derivation of the Protein-Micelle Complex—To define the relative helix orientation and to position the protein on the surface of the micelle, additional “global” restraints were employed. They were (a) HN-N RDCs and (b) two further additional restraints, namely (b1) the hydrophobic moments of the amphipathic helices should point toward the center of the micelle, and (b2) the structure should fit to the geometrical size of the micelle. All calculations in Part II, except in Step 1 (see below), were carried out in X-PLOR and Protein Constructor, a program written by a few of us, 3J. Zdunek, M. Lindgren, M. Karlsson, P. O. Westlund, and S. Wijmenga, manuscript in preparation. which employs calls on XPLOR and aims to smooth structure calculations based on “global” constraints (for a more detailed description, see section iiia). In Step 1, a Monte Carlo simulation is performed to fit for each helix the calculated to the experimental RDCs using the dipolar wave formalism (45Mesleh M.F. Lee S. Veglia G. Thiriot D.S. Marassi F.M. Opella S.J. J. Am. Chem. Soc. 2003; 125: 8928-8935Crossref PubMed Scopus (90) Google Scholar, 46Mascioni A. Veglia G. J. Am. Chem. Soc. 2003; 125: 12520-12526Crossref PubMed Scopus (38) Google Scholar). These Monte Carlo fittings were performed via Matlab scripts using the dipolar wave formulations by Mesleh et al. (45Mesleh M.F. Lee S. Veglia G. Thiriot D.S. Marassi F.M. Opella S.J. J. Am. Chem. Soc. 2003; 125: 8928-8935Crossref PubMed Scopus (90) Google Scholar) and/or by Mascioni et al. (46Mascioni A. Veglia G. J. Am. Chem. Soc. 2003; 125: 12520-12526Crossref PubMed Scopus (38) Google Scholar) and via a procedure incorporated into Protein Constructor, which employs the dipolar wave formulation by Mascioni et al. (46Mascioni A. Veglia G. J. Am. Chem. Soc. 2003; 125: 12520-12526Crossref PubMed Scopus (38) Google Scholar). This resulted in a set of angles (three per helix rigid body: θ, φ, and ρ) that defines the helix orientations in three-dimensional space, albeit with some unavoidable degeneracy intrinsic to derivation of angles from RDCs. This degeneracy is resolved by the geometric requirements resulting from “global” restraints mentioned above. In Step 2, the 10 “best” helix structures of Part I were then supplied to Protein Constructor. Each of the structures was logically divided into fragments detected by the program as helices or joints. Based on the results from Part I, the apoCIII molecule was segmented into six helical regions in the following order: helix 1 (residues 8–18), helix 2 (residues 20–29), helix 3 (residues 33–43), helix 4 (residues 46–54), helix 5 (residues 55–65) and helix 6 (residues 74–79). The rest of the molecule was treated as joints and enumerated in appropriate consecutive order. The average length of each joint between helices was estimated based on the 100 structures calculated in Part I of the structure determination. Those values together with the three angles (φ, θ, and ρ) orienting helices axes in three-dimensional space were submitted to the Protein Constructor as parameters. Having this information and being equipped with the algorithms able to calculate helical axes and hydrophobic moments from the structure, the Protein Constructor appropriately positioned each helix one after another according to the supplied information on the surface of the micelle. The orientations of the short junctions between helices were determined in the first approximation as averages of the orientations of the adjacent helices. Putting the length of the hydrophobic moments equal to the size of the radius of the micelle and requiring the hydrophobic moments to point to the same place in space, we ensured that all helices stayed on the surface of the micelle. In Step 3, the unavoidable disruptions at helical junctions were then “healed” in Protein Constructor via a loop closure algorithm in 200 iterations (47Canutescu A. Dunbrack Jr. R.L. Protein Sci. 2003; 12: 963-972Crossref PubMed Scopus (421) Google Scholar). In Step 4, the resulting structures were then minimized as rigid bodies in Protein Constructor via a call to XPLOR-NIH using positional constraints on Cα atoms (i.e. the Cα atoms of residues 8, 24, 29, 38, 50, 60, and 76 were constrained in space, corresponding to about one Cα atom per helix rigid body). In Step 5, the five lowest energy structures were then refined by XPLOR-NIH via a gentle Cartesian space SA protocol (500 to 25 K, step 25 K) followed by 3000 steps of Powell energy minimization employing all NMR restraints. Each of the five structures generated 10 new final structures. During this refinement, the seven Cα backbone atoms of Step 4 (in the middle or near the middle of each helix) were kept in space to ensure the helices stayed on the micelle. Ad" @default.
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• W2125103697 date "2008-06-01" @default.
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• W2125103697 title "Structure and Dynamics of Human Apolipoprotein CIII" @default.
• W2125103697 cites W111266322 @default.
• W2125103697 cites W1527680278 @default.
• W2125103697 cites W1538092599 @default.
• W2125103697 cites W1589295940 @default.
• W2125103697 cites W1591191431 @default.
• W2125103697 cites W1863056418 @default.
• W2125103697 cites W1963872182 @default.
• W2125103697 cites W1975304761 @default.
• W2125103697 cites W1992820696 @default.
• W2125103697 cites W1994543234 @default.
• W2125103697 cites W1997090264 @default.
• W2125103697 cites W2000948557 @default.
• W2125103697 cites W2001995220 @default.
• W2125103697 cites W2002022989 @default.
• W2125103697 cites W2009316979 @default.
• W2125103697 cites W2010180260 @default.
• W2125103697 cites W2011118302 @default.
• W2125103697 cites W2018203120 @default.
• W2125103697 cites W2021445521 @default.
• W2125103697 cites W2026247887 @default.
• W2125103697 cites W2026582002 @default.
• W2125103697 cites W2027824759 @default.
• W2125103697 cites W2027978582 @default.
• W2125103697 cites W2029164903 @default.
• W2125103697 cites W2029667189 @default.
• W2125103697 cites W2037031239 @default.
• W2125103697 cites W2038290243 @default.
• W2125103697 cites W2039978909 @default.
• W2125103697 cites W2049825478 @default.
• W2125103697 cites W2050945124 @default.
• W2125103697 cites W2052288531 @default.
• W2125103697 cites W2053202752 @default.
• W2125103697 cites W2061144516 @default.
• W2125103697 cites W2066768773 @default.
• W2125103697 cites W2067348020 @default.
• W2125103697 cites W2067484689 @default.
• W2125103697 cites W2078817107 @default.
• W2125103697 cites W2080986054 @default.
• W2125103697 cites W2091242259 @default.
• W2125103697 cites W2094889474 @default.
• W2125103697 cites W2096432979 @default.
• W2125103697 cites W2099356919 @default.
• W2125103697 cites W2100615599 @default.
• W2125103697 cites W2105037825 @default.
• W2125103697 cites W2108657985 @default.
• W2125103697 cites W2111307154 @default.
• W2125103697 cites W2112103709 @default.
• W2125103697 cites W2115816529 @default.
• W2125103697 cites W2117622708 @default.
• W2125103697 cites W2121452195 @default.
• W2125103697 cites W2121861656 @default.
• W2125103697 cites W2123715983 @default.
• W2125103697 cites W2129879061 @default.
• W2125103697 cites W2139240414 @default.
• W2125103697 cites W2154521687 @default.
• W2125103697 cites W2159786214 @default.
• W2125103697 cites W2162588312 @default.
• W2125103697 cites W2169821755 @default.
• W2125103697 cites W2170671445 @default.
• W2125103697 cites W2277423850 @default.
• W2125103697 cites W2401829918 @default.
• W2125103697 cites W4625298 @default.
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