We previously defined a cholesterol recognition/interaction amino acid consensus sequence [CRAC: L/V-X (1-5)-Y-X (1-5)-R/K] in the carboxyl terminus of the peripheral-type benzodiazepine receptor (PBR), a high-affinity drug and cholesterol-binding protein present in the outer mitochondrial membrane protein. This protein is involved in the regulation of cholesterol transport into the mitochondria, the rate-determining step in steroid biosynthesis. Reconstituted wild-type recombinant PBR into proteoliposomes demonstrated high-affinity 2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide and cholesterol binding. In the present work, we functionally and structurally characterized this CRAC motif using reconstituted recombinant PBR and nuclear magnetic resonance. Deletion of the C-terminal domain of PBR and mutation of the highly conserved among all PBR amino acid sequences Y152 of the CRAC domain resulted in loss of the ability of mutant recPBR to bind cholesterol. Nuclear magnetic resonance analysis of a PBR C-terminal peptide (144-169) containing the CRAC domain indicated a helical conformation for the L144-S159 fragment. As a result of the side-chain distribution, a groove that could fit a cholesterol molecule is delineated, on one hand, by Y152, T148, and L144, and, on the other hand, by Y153, M149, and A145. The aromatic rings of Y152 and Y153 assigned as essential residues for cholesterol binding constitute the gate of the groove. Furthermore, the side chain of R156 may cap the groove by interacting with the sterol hydroxyl group. These results provide structural and functional evidence supporting the finding that the CRAC domain in the cytosolic carboxyl-terminal domain of PBR might be responsible for the uptake and translocation of cholesterol into the mitochondria.

Asf1 is a conserved histone chaperone implicated in nucleosome assembly, transcriptional silencing, and the cellular response to DNA damage. We solved the NMR solution structure of the Nterminal functional domain of the human Asf1a isoform, and we identified by NMR chemical shift mapping a surface of Asf1a that binds the C-terminal helix of histone H3. This binding surface forms a highly conserved hydrophobic groove surrounded by charged residues. Mutations within this binding site decreased the affinity of Asf1a for the histone H3͞H4 complex in vitro, and the same mutations in the homologous yeast protein led to transcriptional silencing defects, DNA damage sensitivity, and thermosensitive growth. We have thus obtained direct experimental evidence of the mode of binding between a histone and one of its chaperones and genetic data suggesting that this interaction is important in both the DNA damage response and transcriptional silencing.Asf1 histone chaperone ͉ chromatin ͉ DNA damage ͉ NMR chemical shift mapping ͉ nucleosome assembly D NA in eukaryotic cells is packaged as nucleosome core particles containing Ϸ145 bp of DNA wrapped around an octamer comprised of two copies each of histones H2A, H2B, H3, and H4 (1). Assembly of histones into nucleosomes is a tightly orchestrated process (2, 3). Asf1 is a highly conserved histone chaperone that has been linked to both nucleosome assembly and disassembly (4-7). Asf1 interacts with two functional classes of protein: chromatin components, including histone H3 (8), the Hir proteins (9, 10), and the second subunit of CAF-I (5, 11, 12), and checkpoint kinases, including the Rad53 checkpoint kinase in budding yeast (13,14) and the Tousled-like kinases in metazoans (15). The function of most of these interactions has not been defined. However, a Hir binding region of Asf1 was implicated in telomeric silencing but not required for resistance to genotoxic stress (16). Further work is necessary to determine the functional role of the remaining interactions and, in particular, for defining which Asf1 partners are required for the DNA damage response and for optimal cell growth. In this work, we present the solution structure of the functional Nterminal domain of human Asf1a, and we identify its histone H3 binding site. We show that Asf1 mutants severely defective in histone H3͞H4 binding are incompetent in silencing and in providing resistance to DNA damage. MethodsProtein Production. pETM30 allowed the production of recombinant (His) 6 -GST-Tev site-fusion proteins in Escherichia coli strain BL21 gold (DE3). Unlabeled and uniformly labeled proteins were obtained as described in ref. 17. After Tev cleavage, the 15 N-labeled-H3 (122-135) peptide was further purified by reverse-phase chromatography. The NMR buffer was described in ref.17. An unlabeled peptide spanning the 122-133 sequence of histone H3 was obtained by chemical synthesis (Epytop, Nîmes, France). The protein concentrations were precisely measured by amino acid analysis.NMR Structure Determination and Binding Expe...

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.

Deuterium nuclear magnetic resonance (2H NMR) was used to study the interaction of a cationic amphiphilic peptide with pure DMPC membranes and with mixed bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylserine (DMPS). The choline and serine headgroups were selectively deuteriated at the alpha and beta positions. The amphiphilic peptide, with 20 leucine residues in the hydrophobic core and two cationic hydrophilic lysine residues at each end, spanned the lipid bilayer. Although 2H NMR experiments using DMPC with perdeuteriated fatty acyl chains showed that the average order parameter of the hydrophobic region was not significantly modified by the incorporation of the amphiphilic peptide, for either DMPC or DMPC/DMPS (5:1) bilayers, large perturbations of the quadrupolar splittings of the choline and serine headgroups were observed. The results obtained with the DMPC headgroup suggest that the incorporation of the cationic peptide in both DMPC and DMPC/DMPS (5:1) bilayers leads to a structural perturbation directly related to the net charge on the membrane surface. The magnitude of the observed effect seems to be similar to those observed previously with other cationic molecules [Seelig, J., MacDonald, P.M., & Scherer, P.G. (1987) Biochemistry 26, 7535-7541]. Two of the three quadrupolar splittings of the PS headgroup exhibited large variations in the presence of the amphiphilic peptide, while the third one remained unchanged. Our data have led us to propose a model describing the influence of membrane surface charges on headgroup conformation. In this model, the surface charge is represented as a uniform charge distribution. The electric field due to the charges produces a torque which rotates the polar headgroups.(ABSTRACT TRUNCATED AT 250 WORDS)