Architecture of the 99 bp DNA-six-protein regulatory complex of the lambda att site

The highly directional and tightly regulated recombination reaction used to site-specifically excise the bacteriophage lambda chromosome out of its E. coli host chromosome requires the binding of six sequence-specific proteins to a 99 bp segment of the phage att site. To gain structural insights into this recombination pathway, we measured 27 FRET distances between eight points on the 99 bp regulatory DNA bound with all six proteins. Triangulation of these distances using a metric matrix distance-geometry algorithm provided coordinates for these eight points. The resulting path for the protein-bound regulatory DNA, which fits well with the genetics, biochemistry, and X-ray crystal structures describing the individual proteins and their interactions with DNA, provides a new structural perspective into the molecular mechanism and regulation of the recombination reaction and illustrates a design by which different families of higher-order complexes can be assembled from different numbers and combinations of the same few proteins.     

Induction of reactive oxygen species and cell survival in the presence of advanced glycation end products and similar structures

Advanced glycation end products (AGEs) that arise from the reaction of sugars with protein side chains and the terminal amino group are supposed to be involved in the pathogenesis of several diseases and therefore the effects of AGEs on cells are the objective of numerous investigations. The effects of AGEs on cells are commonly assumed to be transduced via the receptor for AGEs (RAGE) but there are also other receptors known to interact with AGEs and they are likely to be involved in signal transduction. The primary cellular effect of AGEs on cultured cells was found to be the formation of reactive oxygen species (ROS). For the present study one murine and three human cell lines were used. The effects of a set of different highly modified AGEs and AGE-like compounds derived from the incubation of different modifiers with BSA were tested for their effects on these cells. Almost all AGEs tested induced the production of reactive oxygen species (ROS) in the different cell lines although the intensity of the detected signals varied considerably between the cell lines and are strongly dependent on the AGE used for cell activation. The most highly modified BSA-species were shown to inhibit cell growth in all cell lines, whereas a moderately modified glucose derived BSA-AGE and BSA-GA(red) did not show any inhibitory effect on cell growth even when a high ROS formation was detected.     

<Emphasis Type=Italic>Filifactor alocis</Emphasis> - involvement in periodontal biofilms

Background  

Bacteria in periodontal pockets develop complex sessile communities that attach to the tooth surface. These highly dynamic microfloral environments challenge both clinicians and researchers alike. The exploration of structural organisation and bacterial interactions within these biofilms is critically important for a thorough understanding of periodontal disease. In recent years, Filifactor alocis, a fastidious, Gram-positive, obligately anaerobic rod was repeatedly identified in periodontal lesions using DNA-based methods. It has been suggested to be a marker for periodontal deterioration. The present study investigated the epidemiology of F. alocis in periodontal pockets and analysed the spatial arrangement and architectural role of the organism in in vivo grown subgingival biofilms.     

Toward parathyroid hormone minimization: conformational studies of cyclic PTH(1-14) analogues

The N-terminal fragment of PTH(1-34) is critical for PTH1 receptor activation. Various modifications of PTH(1-14) have been shown to result in a considerable increase in signaling potency [Shimizu et al. (2000) J. Biol. Chem. 275, 21836-21843]. Our structural investigations revealed an unusually stable helical structure of the signaling domain (1-14), where residues 6 (Gln) and 10 (Gln or Asn) were located on the same face of the alpha-helix. To test whether a stable N-terminal alpha-helix is required for productive interaction with PTH1 receptor, we designed two conformationally restricted PTH(1-14) analogues, each containing a lactam bridge at positions 6 and 10. Specifically, substitutions Gln(6)-->Glu(6) and Asn(10)-->Lys(10) were introduced into the most potent [Ala(1,3,12),Gln(10),Har(11),Trp(14)]PTH(1-14)NH2 agonist. Both the Glu(6)-Lys(10) and Lys(6)-Glu(10) lactam-bridged analogues were characterized to examine the importance of orientation of the lactam. According to biological studies [Shimizu et al. (2003) Biochemistry 42, 2282-2290], none of the 6/10 substituted analogues (linear or cyclic) remained as active as the parent peptide. However, relative to their corresponding linear peptides, lactam-bridged analogues either maintained potency or showed 6-fold improvement. High-resolution structures as determined by 1H NMR and NOE-restrained molecular dynamics simulations clearly illustrate the structural differences between the linear and cyclic PTH(1-14) fragments, supporting the hypothesis that an alpha-helix is the preferred bioactive conformation of the N-terminal fragment of PTH. In addition, our results demonstrate that the structural order of the very first residues (1-4) of the signaling domain plays a significant role in PTH action.     

The retention factor p11 confers an endoplasmic reticulum-localization signal to the potassium channel TASK-1

The interaction of the adaptor protein p11, also denoted S100A10, with the C-terminus of the two-pore-domain K+ channel TASK-1 was studied using yeast two-hybrid analysis, glutathione S-transferase pull-down, and co-immunoprecipitation. We found that p11 interacts with a 40 amino-acid region in the proximal C-terminus of the channel. In heterologous expression systems, deletion of the p11-interacting domain enhanced surface expression of TASK-1. Attachment of the p11-interacting domain to the cytosolic tail of the reporter protein CD8 caused retention/retrieval of the construct in the endoplasmic reticulum (ER). Attachment of the last 36 amino acids of p11 to CD8 also caused ER localization, which was abolished by removal or mutation of a putative retention motif (H/K)xKxxx, at the C-terminal end of p11. Imaging of EGFP-tagged TASK-1 channels in COS cells suggested that wild-type TASK-1 was largely retained in the ER. Knockdown of p11 with siRNA enhanced trafficking of TASK-1 to the surface membrane. Our results suggest that binding of p11 to TASK-1 retards the surface expression of the channel, most likely by virtue of a di-lysine retention signal at the C-terminus of p11. Thus, the cytosolic protein p11 may represent a 'retention factor' that causes localization of the channel to the ER.     

Optimization of the All-D Peptide D3 for Aβ Oligomer Elimination

The aggregation of amyloid-β (Aβ) is postulated to be the crucial event in Alzheimer’s disease (AD). In particular, small neurotoxic Aβ oligomers are considered to be responsible for the development and progression of AD. Therefore, elimination of thesis oligomers represents a potential causal therapy of AD. Starting from the well-characterized d-enantiomeric peptide D3, we identified D3 derivatives that bind monomeric Aβ. The underlying hypothesis is that ligands bind monomeric Aβ and stabilize these species within the various equilibria with Aβ assemblies, leading ultimately to the elimination of Aβ oligomers. One of the hereby identified d-peptides, DB3, and a head-to-tail tandem of DB3, DB3DB3, were studied in detail. Both peptides were found to: (i) inhibit the formation of Thioflavin T-positive fibrils; (ii) bind to Aβ monomers with micromolar affinities; (iii) eliminate Aβ oligomers; (iv) reduce Aβ-induced cytotoxicity; and (v) disassemble preformed Aβ aggregates. The beneficial effects of DB3 were improved by DB3DB3, which showed highly enhanced efficacy. Our approach yielded Aβ monomer-stabilizing ligands that can be investigated as a suitable therapeutic strategy against AD.     

A conformational comparison of two stereoisomeric cyclic dermorphin analogues employing NMR and computer simulations

In a continuation of our program to study the structure-activity relationship of peptide opiates, we report the conformational analysis of two cyclic tetrapeptides related to dermorphin--Tyr-c[D-Orn-Phe-Asp]-NH2 and Tyr-c[D-Asp-Phe-Orn]-NH2. These analogues have similar binding properties marked by a high selectivity for the mu-opioid receptors because of a drastic decrease in the affinity for the delta-opioid receptor. The conformational preferences of these analogues of dermorphin determined from proton nmr, molecular dynamics, and energy minimizations are quite similar. The constraint of the 13-membered ring formed from cyclization is quite evident from the conformational analysis. The constrained ring system acts as a template maintaining the relative orientation of the exocyclic tyrosine and side chain of phenylalanine. Two intramolecular hydrogen bonds measured for the D-Orn analogue in DMSO were disrupted upon the addition of water. For the D-Asp analogue, two intramolecular hydrogen bonds were found stable in DMSO and water. The global conformations of the two peptides determined from nuclear Overhauser effects did not change with the solvent titration. The difference in the hydrogen bonding within the 13-membered ring may account for the slight differences observed in the efficacy of the analogues at the mu-opioid receptors.     

The mid-region of parathyroid hormone (1-34) serves as a functional docking domain in receptor activation

Elucidating the bimolecular interface between parathyroid hormone (PTH) and its cognate G protein-coupled receptor (PTHR1) should yield insights into the basis of molecular recognition and the mechanism of ligand-mediated intracellular signaling for a system that is critically important in regulating calcium levels in blood. We used photoaffinity scanning (PAS) to identify key ligand-receptor interactions for residues from the unstructured mid-region domain of PTH-(1-34). Four PTH analogues, containing a single photoreactive p-benzoylphenylalanine (Bpa) residue in position 11, 15, 18, or 21, were found to photo-cross-link within receptor regions [165-176], [183-189], [190-298], and [165-176], respectively. Addition of these mid-region contacts as constraints to our previously proposed model of the PTH-PTHR1 complex and extensive molecular simulation experiments enables substantial refinement of the model. Specifically, (1) the overall receptor-bound conformation of the hormone is not extended, but bent; (2) helix [169-176] of the N-terminal extracellular domain (N-ECD) of the receptor is redirected toward the heptahelical bundle; and (3) the hormone traverses between the top of transmembrane (TM) helices 1 and 2, rather than between TM-7 and TM-1. This significantly alters the model of both the receptor-bound tertiary structure of the hormone and the topological orientation of the C-terminus of the N-ECD in the hormone-receptor bimolecular complex. We propose that the mid-region of PTH-(1-34) has a role in fixing, by extensive contacts with the receptor, the entry of the N-terminal helix of the hormone into the heptahelical bundle between TM-1 and TM-2. This anchorage would orient the amino terminus into position to activate the receptor.     

Structural analysis of the human cannabinoid receptor one carboxyl‐terminus identifies two amphipathic helices

Recent research has implicated the C‐terminus of G‐protein coupled receptors in key events such as receptor activation and subsequent intracellular sorting, yet obtaining structural information of the entire C‐tail has proven a formidable task. Here, a peptide corresponding to the full‐length C‐tail of the human CB1 receptor (residues 400–472) was expressed in E.coli and purified in a soluble form. Circular dichroism (CD) spectroscopy revealed that the peptide adopts an α‐helical conformation in negatively charged and zwitterionic detergents (48–51% and 36–38%, respectively), whereas it exhibited the CD signature of unordered structure at low concentration in aqueous solution. Interestingly, 27% helicity was displayed at high peptide concentration suggesting that self‐association induces helix formation in the absence of a membrane mimetic. NMR spectroscopy of the doubly labeled (¹⁵N‐ and ¹³C‐) C‐terminus in dodecylphosphocholine (DPC) identified two amphipathic α‐helical domains. The first domain, S401‐F412, corresponds to the helix 8 common to G protein‐coupled receptors while the second domain, A440‐M461, is a newly identified structural motif in the distal region of the carboxyl‐terminus of the receptor. Molecular modeling of the C‐tail in DPC indicates that both helices lie parallel to the plane of the membrane with their hydrophobic and hydrophilic faces poised for critical interactions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 565–573, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Insights into succession processes using temporally repeated habitat models: results from a long‐term study in a post‐mining landscape

Questions: The early phases of primary succession are governed by chance events and dispersal‐related processes in an environment that is largely free of competition. Thus, the predictability of vegetation patterns using environmental site factors can be expected to be low and spatial autocorrelation to be high. We asked whether the match between vegetation and environment becomes better in the course of succession, and whether vegetation types shift their realized niche with time. Location: Lignite mining region in Central Germany, the post‐mining landscape “Goitzsche” (Saxony‐Anhalt). Methods: Vegetation types were mapped in a 10‐m grid (total area 4.8 ha), starting in 1995, at 3‐year intervals until 2007. We used a temporal comparison of habitat models. We applied: GLS regression to partition the variation in coverage of vegetation types into environmental (soil pH) and spatial components; logistic regression to model the presence/absence of vegetation types along a soil acidity gradient; and autologistic regression allowing for soil acidity and neighbourhood effects. Results: For most vegetation types, the proportion of variation explained by space was high but declined during succession. The outcome of autologistic models suggests that soil acidity often plays a minor role compared to neighbourhood effects in the earlier phase of succession than 12 years later. For four vegetation types, the pH range in which the type was expected to be dominant clearly became smaller with time. These trends support the view that the role of processes related to chance and dispersal decrease with time, while those related to environmental filtering mediated by biotic interactions increase. Conclusions: We conclude that temporal comparisons of spatially explicit habitat models provide insights into changing biotic community processes and their effects on habitat specificity of species or their communities. Thus, this approach may be particularly important for analysis of ecological systems that are not in equilibrium with their environmental drivers.