Solution structure and orientation of the transmembrane anchor domain of the HIV-1-encoded virus protein U by high-resolution and solid-state NMR spectroscopy.

The structure of the membrane anchor domain (VpuMA) of the HIV-1-specific accessory protein Vpu has been investigated in solution and in lipid bilayers by homonuclear two-dimensional and solid-state nuclear magnetic resonance spectroscopy, respectively. Simulated annealing calculations, using the nuclear Overhauser enhancement data for the soluble synthetic peptide Vpu1-39 (positions Met-1-Asp-39) in an aqueous 2,2,2-trifluoroethanol (TFE) solution, afford a compact well-defined U-shaped structure comprised of an initial turn (residues 1-6) followed by a linker (7-9) and a short helix on the N-terminal side (10-16) and a further longer helix on the C-terminal side (22-36). The side chains of the two aromatic residues (Trp-22 and Tyr-29) in the longer helix are directed toward the center of the molecule around which the hydrophobic core of the folded VpuMA is positioned. As the observed solution structure is inconsistent with the formation of ion-conductive membrane pores defined previously for VpuMA in planar lipid bilayers, the isolated VpuMA domain as peptide Vpu1-27 was investigated in oriented phospholipid bilayers by proton-decoupled 15N cross polarization solid-state NMR spectroscopy. The line widths and chemical shift data of three selectively 15N-labeled peptides are consistent with a transmembrane alignment of a helical polypeptide. Chemical shift tensor calculations imply that the data sets are compatible with a model in which the nascent helices of the folded solution structure reassemble to form a more regular linear alpha-helix that lies parallel to the bilayer normal with a tilt angle of

Birch pollen profilin: structural organization and interaction with poly-(L-proline) peptides as revealed by NMR.

The secondary structure of birch pollen profilin, a potent human allergen, was elucidated by multidimensional nuclear magnetic resonance (NMR), as a prerequisite to study the interaction of this profilin with ligands for its poly-(L-proline) (PLP)-binding site. The chemical shifts of the 15N-labeled backbone amide groups were used to monitor complex formation with various PLP peptides. Titration with deca-L-proline (P10) yielded a KD of 0.2 mM. P8 was the shortest PLP to provoke a significant reaction. (GP5)3G bound significantly, confirming the interaction between profilins and the protein VASP containing this motif. Birch profilin interacted also with GP6GP5, found in the cyclase-associated protein (CAP), a suspected profilin ligand.

Solution structure of the cytoplasmic domain of the human immunodeficiency virus type 1 encoded virus protein U (Vpu).

The HIV-1-specific Vpu protein is an 81 amino acid class I integral membrane phosphoprotein that induces degradation of the virus receptor CD4 in the endoplasmic reticulum and enhances the release of virus particles from infected cells. Vpu is of amphipathic nature and consists of a hydrophobic N-terminal membrane anchor proximal to a polar C-terminal cytoplasmic domain. In our recent work, focussed on the structural analysis of the cytoplasmic tail, we established an alpha-helix-flexible-alpha-helix-turn model. Now we present the experimental solution structure of the Vpu cytoplasmic domain which has been elucidated in aqueous 50% trifluoroethanol solution by 2D 1H NMR spectroscopy, and restrained molecular dynamics and energy minimization calculations. Under these conditions the peptide, Vpu32-81, is predominantly monomeric and adopts a well defined helix-interconnection-helix-turn conformation, in which the four regions are bounded by residues 37-51, 52-56, 57-72 and 73-78. The presence of the cis isomer of Pro-75 manifests itself as a doubling of cross peaks of neighbouring residues in the 2D spectra. A related variant peptide, Vpum32-81, in which the Vpu-phosphoacceptor sites Ser52 and Ser56 were exchanged for Asn, adopts a very similar structure and, taken together, provides evidence that the second helix and the turn form a comparatively rigid region. Both helices are amphipathic in character, but show different charge distributions. In general the cytoplasmic region is N-terminally positively charged, passes through a region of alternating charges in helix 1 and then becomes negatively charged. The flexibility of the interconnection permits orientational freedom of the two helices. The motif found here is the first experimentally refined solution structure of the cytoplasmic domain of Vpu, and it is conceivable that these alpha-helices are important for a previously defined physical interaction with an alpha-helical Vpu-responsive element located within the cytoplasmic tail of CD4.

Solution structure of the hydrophilic region of HIV-1 encoded virus protein U (Vpu) by CD and 1H NMR spectroscopy.

The HIV-1 specific Vpu is a class I oligomeric membrane phosphoprotein of unknown structure and mechanism. The first experimental evidence for the position of secondary structural elements present in the hydrophilic C-terminal region of Vpu under various solution regimes is reported. CD data for nine overlapping 15 amino-acid fragments and 3 longer fragments indicate the presence of only transitory amounts of stable structure in aqueous solution alone, while with increasing trifluoroethanol content limiting structures were found indicating two helical segments in the hydrophilic region of Vpu. These limiting structures were more precisely defined from a detailed study of Vpu41-58, Vpu52-74 and Vpu63-81, by a combination of 2D 1H NMR spectroscopy, distance geometry, and restrained molecular dynamics and energy minimization calculations. Sets of low-energy conformations compatible with the quantitative NOE data indicate that Vpu41-58 has an alpha-helix from residues 42 to 50 while a second helix is found for Vpu52-74 from residues 57 to 69. Vpu63-81 shows only the presence of a single reverse turn at residues 74 to 77, without any evidence of helix, under the same conditions. From CD measurements the first helix extends back to residue 30 and is connected to the N-terminal anchor of Vpu. Thus the hydrophilic region of Vpu consists of two alpha-helices joined by a flexible region of 6 or 7 residues, which contains the phosphoacceptor sites of Vpu at positions 52 and 56. The second helix is followed by a single reverse turn and a flexible C-terminus.

The structure of human parathyroid hormone from a study of fragments in solution using 1H NMR spectroscopy and its biological implications.

In order to gain insight into the structure of human parathyroid hormone (hPTH), four fragments [hPTH(1-34), hPTH(18-48), hPTH(28-48), and hPTH(53-84)], which cover all regions of the intact hormone, have been investigated by CD and NMR spectroscopy in combination with distance geometry, and restrained molecular dynamics and energy minimization calculations, under a variety of solution conditions. Significantly, all fragments showed little propensity to form stable structures in aqueous solution alone, and it was only on the addition of trifluoroethanol (TFE) that defined structural features were observed. In an extension of earlier work [Klaus et al. (1991) Biochemistry 30, 6936-6942], hPTH(1-34) in 70% trifluoroethanol (TFE) showed two helices that were longer than in 10% TFE, but essentially showed the same characteristics. Although overlap in the 1H NMR spectra prevented the determination of quantitative NOE data for residues 26-30, the combination of the alpha-proton chemical shift data and quantitative NOE data indicated the helices extend from residues 3 to 13 and 15 to 29. No evidence was found for interaction of the two helical regions. The nature and extent of this second helix in the intact hormone were better defined from the data for hPTH(18-48). Under limiting solution conditions, where the fragment assumed its maximum helical content, a well-defined helix was observed between residues 21 and 38 with a possible discontinuity between Leu-28 and Gln-29. There was little evidence of any form of secondary structure between Gly-38 and the terminus of this fragment, Ser-48. In keeping with this result, the shorter fragment, hPTH(28-48), showed little evidence of stable secondary structure on addition of TFE. From the alpha-proton chemical shifts residues 23-27 appeared to sustain helical structure more readily than the rest of molecule under all solution regimes in both hPTH(1-34) and hPTH(18-48). In contrast to the other two longer fragments hPTH(53-84) showed little propensity for helical secondary structure even at the highest TFE concentrations. However, there was evidence that the molecule did adopt a defined three-dimensional structure. Various long-range NOE's were observed in 10% TFE that allowed the calculation of an open tertiary structure consisting of an initial series of turns surrounded by a loop structure of several loose turns.(ABSTRACT TRUNCATED AT 400 WORDS)