The structures of functional peptides corresponding to the predicted channel-lining M2 segments of the nicotinic acetylcholine receptor (AChR) and of a glutamate receptor of the NMDA subtype (NMDAR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. Both M2 segments form straight transmembrane alpha-helices with no kinks. The AChR M2 peptide inserts in the lipid bilayer at an angle of 12 degrees relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminal side of the membrane, which is assigned to be intracellular. A model built from these solid-state NMR data, and assuming a symmetric pentameric arrangement of M2 helices, results in a funnel-like architecture for the channel, with the wide opening on the N-terminal intracellular side.
The stabilization of p53 against Mdm2-mediated degradation is an important event in DNA damage response. Initial models of p53 stabilization focused on posttranslational modification of p53 that would disrupt the p53-Mdm2 interaction. The N-terminal regions of both p53 and Mdm2 are modified in vivo in response to cellular stress, suggesting that modifications to Mdm2 also may affect the p53-Mdm2 interaction. Our NMR studies of apo-Mdm2 have found that, in addition to Mdm2 residues 25-109 that form the well ordered p53-binding domain that was observed in the p52-Mdm2 complex, Mdm2 residues 16 -24 form a lid that closes over the p53-binding site. The Mdm2 lid, which is strictly conserved in mammals, may help to stabilize apo-Mdm2. It also competes weakly with peptidic and nonpeptidic antagonists. Modifications to the Mdm2 lid may disrupt p53-Mdm2 binding leading to p53 stabilization. Mdm2 and Mdm4 possess nearly identical p53-binding domains but different lids suggesting that lid modifications may select for p53 binding.T he p53 tumor suppressor protein plays an important role in maintaining genome stability and in preventing the development of cancer. In response to various stress signals, p53 activation leads to cell-cycle arrest, apoptosis, or DNA repair (1). p53 is also a transcription factor for mdm2, the product of which negatively regulates both p53 stability and activity (2, 3). Mdm2 affects p53 activity by binding the N-terminal transactivation domain, blocking transcription (4-6). Mdm2 affects p53 stability by targeting it for ubiquitin-dependent degradation (7-9). In normal cells, p53 activity is kept low by Mdm2 (10). In response to DNA damage, p53 is activated by disrupting Mdm2 association and stabilized against Mdm2-dependent degradation (11). p53 activation and stabilization likely are achieved by posttranslational modifications (12); known modifications to p53 include phosphorylation, acetylation, and ubiquitination (1, 12). In one example of p53 stabilization by posttranslational modification, the phosphorylation of p53 nuclear export signals (13-15) blocks export and leads to p53 accumulation in the nucleus. Other details, particularly concerning p53 activation, are unclear.One model of p53 activation is to disrupt the p53-Mdm2 interaction by phosphorylation (16). p53 is heavily phosphorylated after DNA damage (17-19). Several p53 residues at the p53-Mdm2 interface have been identified as targets or potential targets for kinases that are activated in response to DNA damage (20)(21)(22)(23)(24)(25)(26). Whereas in vitro phosphorylation of some of these residues can be shown qualitatively to weaken p53-Mdm2 association, quantitative studies have determined that phosphorylation of single p53 sites (that are known substrates) has no effect on Mdm2 binding (27)(28)(29). These results are consistent with studies in which single and multiple replacement of phosphorylatable p53 residues with alanine had no affect on p53 activity (3,(30)(31)(32). These in vivo results suggest that single-site p53 phospho...
Solid-state NMR spectroscopy was used to determine the orientations of two amphipathic helical peptides associated with lipid bilayers. A single spectral parameter provides sufficient orientational information for these peptides, which are known, from other methods, to be helical. The orientations of the peptides were determined using the 15N chemical shift observed for specifically labeled peptide sites. Magainin, an antibiotic peptide from frog skin, was found to lie in the plane of the bilayer. M2 delta, a helical segment of the nicotinic acetylcholine receptor, was found to span the membrane, perpendicular to the plane of the bilayer. These findings have important implications for the mechanisms of biological functions of these peptides.
Magainin2 is a 23-residue antibiotic peptide that disrupts the ionic gradient across certain cell membranes. Two-dimensional 1H NMR spectroscopy was used to investigate the structure of the peptide in three of the membrane environments most commonly employed in biophysical studies. Sequence-specific resonance assignments were determined for the peptide in perdeuterated dodecylphosphocholine (DPC) and sodium dodecylsulfate micelles and confirmed for the peptide in 2,2,2-trifluoroethanol solution. The secondary structure is shown to be helical in all of the solvent systems. The NMR data were used as a set of restraints for a simulated annealing protocol that generated a family of three-dimensional structures of the peptide in DPC micelles, which superimposed best between residues 4 and 20. For these residues, the mean pairwise rms difference for the backbone atoms is 0.47 +/- 0.10 A from the average structure. The calculated peptide structures appear to be curved, with the bend centered at residues Phe12 and Gly13.
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