The solution structure of the porcine gastrointestinal peptide hormone motilin was determined in the presence of sodium dodecyl sulfate (SDS) micelles at 28 degrees C using 1H nuclear magnetic resonance, full relaxation matrix analysis, and structure calculations based on restrained molecular dynamics. The structure of motilin in SDS micelles is described by a reverse gamma-turn and a beta-turn of type II in the N terminal end, an alpha-helical region in the middle of the molecule, and an extended structure at the C terminus. The position of the motilin molecule relative to the SDS micelles was probed by adding spin-labeled stearic acids, containing 12-doxyl or 5-doxyl spin-labels. We observed selective broadening of the proton resonances of residues 3-5 and concluded that they must be located in the interior of the micelle. These experiments suggest a structural model in which the hydrophobic N terminus consists of two well-defined turns buried in the interior of the micelle, whereas the amphiphilic alpha-helical part is located at the surface of the micelle. Spectral density mapping using a 13C label on the alphaC of Leu10 gave overall rotational correlation times taum of 6.6 and 4.5 ns at 35 and 45 degrees C, respectively. The long correlation time in combination with a high order parameter (S = 0.92) indicates that motilin has a rigid structure in the complex with the SDS micelle.

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 lipidbound 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 ϳ10-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 2 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 (1-3). The apolipoproteins regulate blood lipid levels by interaction with specialized endocytotic receptors and by modulating enzyme activities and lipid exchange reactions (4). 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 (2-4). 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) (4, 5). 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 (2, 4, 6). Overexpression of apoCIII in transgenic mice leads to severely increased plasma triglyceride levels due to accumulation of very low density lipoprotein-like lipoprotein remnants w...

Hepatitis B virus (HBV) replication is initiated by HBV RT binding to the highly conserved encapsidation signal, epsilon, at the 5′ end of the RNA pregenome. Epsilon contains an apical stem–loop, whose residues are either totally conserved or show rare non-disruptive mutations. Here we present the structure of the apical stem–loop based on NOE, RDC and 1H chemical shift NMR data. The 1H chemical shifts proved to be crucial to define the loop conformation. The loop sequence 5′-CUGUGC-3′ folds into a UGU triloop with a CG closing base pair and a bulged out C and hence forms a pseudo-triloop, a proposed protein recognition motif. In the UGU loop conformations most consistent with experimental data, the guanine nucleobase is located on the minor groove face and the two uracil bases on the major groove face. The underlying helix is disrupted by a conserved non-paired U bulge. This U bulge adopts multiple conformations, with the nucleobase being located either in the major groove or partially intercalated in the helix from the minor groove side, and bends the helical stem. The pseudo-triloop motif, together with the U bulge, may represent important anchor points for the initial recognition of epsilon by the viral RT.

Apolipoprotein CII (apoCII), a surface constituent of plasma lipoproteins, is the activator for lipoprotein lipase (LPL) and is therefore central for lipid transport in blood. The three-dimensional structure of (13)C-, (15)N-enriched human full-length apoCII in complex with sodium dodecyl sulfate (SDS) micelles is reported. In addition to the structure determination, (15)N-relaxation measurements have been performed at two magnetic fields to characterize the dynamics of the backbone of apoCII in the complex. The relaxation data also provided global structural constraints, viz. the orientation of helices in the complex. In addition, global constraints were derived from the fact that apoCII helices are attached to the surface of the SDS micelle and that the hydrophobic moments of each helix faces the interior of the micelle. These three categories of global constraints, together with the local classical NMR constraints, were sufficient to define the 3D structure of the apoCII-SDS micelle complex. To our knowledge, this presents the first example in which the global structure of a protein-SDS micelle complex has been determined. The C-terminal helix of apoCII is known to be responsible for the activation of LPL. This helix is distinguished from the other helices by a higher degree of internal motion on the nanosecond time scale as shown by the relaxation data. The overall structure and the internal dynamics, combined with previous mutation data, give important clues toward a possible mechanism for the activation of LPL by apoCII.

NMR spectroscopy in aqueous and dimethyl sulfoxide/water solutions is used to determine the three-dimensional structures of microcystin-LR, a cyclic cyanobacterial heptapeptide toxin which is a potent inhibitor of type 1 and type 2A protein phosphatases. The conformations of this toxic peptide are studied using a simulated annealing (SA) protocol followed by refined SA calculations in vacuo and free MD simulations in water. Only one conformational family in each solvent is found. The peptide ring has a saddle-shaped form, essentially the same in both solvents. The structural difference observed between the two solution structures is located to the part consisting of Mdha, Ala, and Leu. This peptide segment is not present in nodularin, a cyclic pentapeptide of similar toxicity. The Arg side chain is very flexible, while the side chain of Leu is well defined. The side chain of Adda, essential for toxicity, is constrained in the vicinity of the backbone ring but appears to be flexible in the more remote part.