Conformational flexibility in the apolipoprotein E amino‐terminal domain structure determined from three new crystal forms: Implications for lipid binding

Abstract

An amino-terminal fragment of human apolipoprotein E3~residues 1-165! has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic~P2 1 2 1 2 1 ! crystal form of the amino-terminal 22 kDa fragment of apolipoprotein E~residues 1-191!. A second orthorhombic crystal form~P2 1 2 1 2 1 with cell dimensions differing from the first form! and a trigonal~P3 1 21! crystal form were also characterized. The structures of the first orthorhombic and the trigonal form were determined by seleno-methionine multiwavelength anomalous dispersion, and the structure of the second orthorhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high-quality electron-density maps, which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four-helix bundle and a number of multiconformation side chains. The three crystal forms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this antiparallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four-helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association.

Versatility in ligand recognition by LDL receptor family proteins: advances and frontiers

“…Davis CG, Goldstein A. Four helix bundle structure of the N-terminal domain of apoE in its lipid-free form (pdb ID code 1BZ4; [34]). Ribbons representing the four helices in the N-terminal domain are colored red (helix 1), blue (helix 2), green (helix 3) and yellow (helix 4), and ribbons of the connecting regions are colored gray.…”

Structure and Dynamics of Human Apolipoprotein CIII

“…The NMR sample of apoCIII in complex with SDS micelles was prepared from lyophilized 15 N/ 13 C-labeled apoCIII as described (23), leading to the final ApoCIII-NMR sample: ϳ0.5 mM apoCIII, 180 mM SDS-d 25 , 8% D 2 O, 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 (23).…”

“…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 15 N/ 1 H HSQC experiments. Prior to each experiment, the temperature was calibrated using tetramethylammonium.…”

“…The RDC data on the M13 coat protein were measured in a similar fashion. 15 MHz. The data points were recorded in an interleaved manner to minimize effects of spectrometer drift and temperature fluctuations.…”

“…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, 13 C chemical shift-derived backbone dihedral angles, 3 J HNH␣ couplings, NMR multiple-field 15 N-spin backbone relaxation, and 15 N/ 1 H 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.…”