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A G‐rich sequence found in the regulatory region of the RANKL gene, which is associated with homeostasis of bone metabolism, folds into a two‐quartet basket‐type G‐quadruplex stabilized by A⋅G⋅A and G⋅G⋅G base‐triads. Perusal of local structural features together with G/A‐to‐T modifications uncovered the critical role of A5 for the formation of a distinct antiparallel two‐quartet topology and not the three‐quartet topology that would be expected based on the sequence with four GGG‐tracts alone. The structural changes induced by the A5‐to‐T5 modification include a switch in orientation and relative positions of G‐strands that together with anti to syn reorientation of G12 provide insights into the complexity of the interactions that influence the folding of G‐rich DNA. Understanding the impact of loop residues on the stability and formation of G‐quadruplexes advances our knowledge and ability to predict structures adopted by G‐rich sequences, which are involved in regulatory mechanisms in the cell, and may also facilitate drug design. [ABSTRACT FROM AUTHOR]

In this study, we report the first atomic resolution structure of a stable G-hairpin formed by a natively occurring DNA sequence. An 11-nt long G-rich DNA oligonucleotide, 5'-d(GTGTGGGTGTG)-3', corresponding to the most abundant sequence motif in irregular telomeric DNA from Saccharomyces cerevisiae (yeast), is demonstrated to adopt a novel type of mixed parallel/antiparallel fold-back DNA structure, which is stabilized by dynamic G:G base pairs that transit between N1-carbonyl symmetric and N1-carbonyl, N7-amino base-pairing arrangements. Although the studied sequence first appears to possess a low capacity for base pairing, it forms a thermodynamically stable structure with a rather complex topology that includes a chain reversal arrangement of the backbone in the center of the continuous G-tract and 3'-to-5' stacking of the terminal residues. The structure reveals previously unknown principles of the folding of G-rich oligonucleotides that could be applied to the prediction of natural and/or the design of artificial recognition DNA elements. The structure also demonstrates that the folding landscapes of short DNA single strands is much more complex than previously assumed. [ABSTRACT FROM AUTHOR]

S100A1 is a member of the Ca2+-binding S100 protein family. It is expressed in brain and heart tissue, where it plays a crucial role as a modulator of Ca2+ homeostasis, energy metabolism, neurotransmitter release, and contractile performance. Biological effects of S100A1 have been attributed to its direct interaction with a variety of target proteins. The (patho)physiological relevance of S100A1 makes it an important molecular target for future therapeutic intervention. S-Nitrosylation is a post-translational modification of proteins, which plays a role in cellular signal transduction under physiological and pathological conditions. In this study, we confirmed that S100A1 protein is endogenously modified by Cys85 S-nitrosylation in PC12 cells, which are a well established model system for studying S100A1 function. We used isothermal calorimetry to show that S-nitrosylation facilitates the formation of Ca2+-loaded S100A1 at physiological ionic strength conditions. To establish the unique influence of the S-nitroso group, our study describes high resolution three-dimensional structures of human apo-S100A1 protein with the Cys85 thiol group in reduced and S-nitrosylated states. Solution structures of the proteins are based on NMR data obtained at physiological ionic strength. Comparative analysis shows that S-nitrosylation fine tunes the overall architecture of S100A1 protein. Although the typical S100 protein intersubunit four-helix bundle is conserved upon S-nitrosylation, the conformation of S100A1 protein is reorganized at the sites most important for target recognition (i.e. the C-terminal helix and the linker connecting two EF-hand domains). In summary, this study discloses cysteine S-nitrosylation as a new factor responsible for increasing functional diversity of S100A1 and helps explain the role of S100A1 as a Ca2+ signal transmitter sensitive to NO/redox equilibrium within cells. [ABSTRACT FROM AUTHOR]

Bone remodeling is a fine-tuned process principally regulated by a cascade triggered by interaction of receptor activator of NF-κB (RANK) and RANK ligand (RANKL). Excessive activity of the RANKL gene leads to increased bone resorption and can influence the incidence of osteoporosis. Although much has been learned about the intracellular signals activated by RANKL/RANK complex, significantly less is known about the molecular mechanisms of regulation of RANKL expression. Here, we report on the structure of an unprecedented DNA G-quadruplex, well-known secondary structure-mediated gene expression regulator, formed by a G-rich sequence found in the regulatory region of a RANKL gene. Solution-state NMR structural study reveals the formation of a three-layered parallel-type G-quadruplex characterized by an unique features, including a G-A bulge. Although a guanine within a G-tract occupies syn glycosidic conformation, bulge-forming residues arrange in a pseudo-loop conformation to facilitate partial 5/6-ring stacking, typical of G-quadruplex structures with parallel G-tracts orientation. Such distinctive structural features protruding from the core of the structure can represent a novel platform for design of highly specific ligands with anti-osteoporotic function. Additionally, our study suggests that the expression of RANKL gene may be regulated by putative folding of its G-rich region into non-B-DNA structure(s). [ABSTRACT FROM AUTHOR]

Spezifische Schleifen‐Interaktionen unter Beteiligung eines Adeninrestes können den Faltungsweg und die Struktur eines G‐Quadruplexes entscheidend beeinflussen. J. Plavec und Mitarbeiter zeigen in ihrer Zuschrift auf S. 15621 ff., dass eine Sequenz mit vier GGG‐Abschnitten in einen G‐Quadruplex mit zwei Quartetts stabilisiert durch A⋅G⋅A‐ und G⋅G⋅G‐Triaden faltet. Modifizierung eines einzelnen Adenins moduliert die Schleifen‐Interaktionen und führt zu ungewöhnlichen Strukturänderungen, einschließlich eines Strukturübergangs von Zweier‐ zu Dreier‐Quartett. [ABSTRACT FROM AUTHOR]

Specific loop interactions involving an adenine residue can critically influence the folding pathway and structure of a G‐quadruplex. In their Communication on page 15395 ff., J. Plavec and co‐workers show that a sequence with four GGG‐tracts folds into a G‐quadruplex with two quartets stabilized by A⋅G⋅A and G⋅G⋅G base‐triads. Modification of a single adenine modulates the loop interactions and leads to exciting structural changes including a two‐to‐three‐quartet structural switch. [ABSTRACT FROM AUTHOR]

Herein we show how sequences that can form different non-canonical structures affect gene expression levels when inserted in the core of σ70-dependent promoter, between the -35 and -10 elements recognized by RNA polymerase, in E. coli. We note that influence on level of GFP expression varies considerably depending on introduction of non-canonical structural elements in the antisense and sense strands as well as with their propensities to form G-triplex, G-hairpin, hairpin or G-quadruplex structures. Moreover, the extent of repression of expression does not relate to the in vitro thermal stability in a simple manner. Repression is most likely caused by steric interference rather than improper distance between the -35 and -10 elements. Although properties like thermal stability and topology can be somewhat different under in vivo and in vitro conditions, our results suggest that the extent of expression suppression cannot be dependent solely on thermal stabilities of Grich structures alone. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a mammalian pathogen that causes gastroenteritis, miscarriages and infections of the central nervous system in immunocompromised individuals. Its main virulence factor is listeriolysin O (LLO), a pore-forming cholesterol-dependent cytolysin (CDC), which enables bacterial escape from the phagolysosome and contributes to bacterial pathogenicity. Details of cholesterol (Chol) recognition and membrane binding mechanisms by LLO are still not known. Here we used 19 F-NMR spectroscopy in order to assess LLO-Chol interactions in solution and in a Chol-rich membrane environment. LLO has six tryptophan residues located in the region of the molecule that is first in contact with lipid membranes. 19 F-LLO, which contained 5-fluoro-tryptophans, was prepared by using isotopic labelling in an E. coli expression system. Signals in the 19 F-NMR spectrum of 19 F-LLO were unambiguously assigned by using a series of single Trp → Phe point mutations. The results employing various cholesterol preparations in solution indicate that tryptophan residues are not directly involved in Chol binding in solution. However, significant chemical shift changes were observed upon LLO binding to Chol-rich membranes, highlighting the role of tryptophan residues in membrane interactions (W512) and oligomerisation (W189 and W489).

Study of interaction of mannose-based ligands with receptor DC-SIGN using high resolution NMR in combination with molecular modelling showed that four α-d-mannoside ligands interact with the binding site predominantly through the mannose moiety. The other two aromatic groups that are bound to α-d-mannose through a glycerol linker demonstrate interaction that can be related to their substitution pattern. Ligand with naphthyl and meta-substituted phenyl ring exhibited the most favourable binding characteristics. In addition to the predicted hydrophobic interactions of aromatic moieties our results propose new contacts of substituted phenyl moiety in the more polar area of the flat binding site of DC-SIGN and thus offer new possibilities in further designing of novel, more potent DC-SIGN antagonists.