NMR Structural Studies Research Articles

NMR structure and functional studies of the fourth FAS1 domain of human periostin

Periostin is a matricellular protein that consists of several structural and functional domains, including EMILIN-like, four internal repeat fasciclin1 (FAS1) domains, and a carboxyl-terminal variable domain. It is known that periostin is associated with various fundamental biological processes and diseases, such as several types of cancer and chronic inflammatory diseases. Despite its important roles, the biological function of each domain is poorly understood. In this study, we expressed the fourth FAS1 (FAS1 IV) domain of human periostin, which was highly soluble and stable enough for structural and functional studies. The three-dimensional structure of FAS1 IV was determined using 3D NMR spectroscopy. The overall structure of FAS1 IV consists of six α-helices, one 310 helix, and eight β-strands. Two triangular α-helical modules formed by three α-helices each are located on one side of the molecule, while the orthogonal β-sheet sandwich module of FAS1 IV is located on the other side. The isolated FAS1 IV domain exhibited cell invasion, migration, and adhesion activities for cancer cell lines comparable to those of the full FAS1 I–IV domain. In conclusion, we propose that the FAS1 IV domain is functionally active in human periostin and provides valuable information for understanding the biological function of periostin.

Nonsymmetrically Substituted 1,1'-Biphenyl-Based Small Molecule Inhibitors of the PD-1/PD-L1 Interaction.

Therapeutic antibodies directed against either programmed cell death-1 protein (PD-1) or its ligand PD-L1 have demonstrated efficacy in the treatment of various cancers. In contrast with antibodies, small molecules have the potential for increased tissue penetration; better pharmacology; and therefore, improved antitumor activity. A series of nonsymmetric C2 inhibitors were synthesized and evaluated for PD-1/PD-L1 interaction inhibition. These compounds induced PD-L1 dimerization and effectively blocked PD-L1/PD-1 interaction in a homogeneous time-resolved fluorescence (HTRF) assay with most inhibitors exhibiting IC50 values in the single-digit nM range and below. Their high inhibitory potency was also demonstrated in a cell-based coculture PD-1 signaling assay where 2 exhibited an EC50 inhibitory activity of 21.8 nM, which approached that of the PD-L1 antibody durvalumab (EC50 = 0.3-1.8 nM). Structural insight into how these inhibitors interact with PD-L1 was gained by using NMR and X-ray cocrystal structure studies. These data support further preclinical evaluation of these compounds as antibody alternatives.

Feasibility of Domain Segmentation of B19V VP1u Using Intein Technology for Structural Studies.

Parvovirus B19 (B19V) is a human pathogen, and the minor capsid protein of B19V possesses a unique N terminus called VP1u that plays a crucial role in the life cycle of the virus. The objective of this study was to develop a method for domain segmentation of B19 VP1u using intein technology, particularly its receptor binding domain (RBD) and phospholipase A2 (PLA2) domain. RBD and PLA2 domains of VP1u were each fused to the DnaE split inteins derived from the Nostoc punctiforme. Each of these precursor proteins was expressed in E. coli. Combining the purified precursors in equal molar ratios resulted in the formation of full-length VP1u. Furthermore, Circular Dichroism (CD) spectroscopy and PLA2 assays were used to probe the structure and activity of the newly formed protein. The CD spectrum of the full length VP1u confirmed the secondary structure of protein, while the PLA2 assay indicated minimal disruption in enzymatic activity. This method would allow for the selective incorporation of NMR-active isotopes into either of the VP1u domains, which can reduce signal overlap in NMR structural determination studies.

NMR Structural Study of Syndecan-4 Transmembrane Domain with Cytoplasmic Region

Syndecan-4 (SDC4) consists of transmembrane heparan sulfate proteoglycan (HSPG) belonging to the syndecan family. It is present in most cell types of Mammalia. Its structure contains a heparan-sulfate-modified extracellular domain, a single transmembrane domain, and a short C-terminal cytoplasmic domain. Regarding the overall cellular function of SDC4, other cells or ligands can bind to its ecto-domain. In addition, 4,5-bisphosphate phosphatidylinositol (PIP2) or protein kinase Cα can bind to its cyto-domain to activate downstream signaling pathways. To understand the signal transduction mechanism of syndecan, it is important to know the interactions between their actual structure and function in vivo. Therefore, it is important to identify the structure of SDC4 to understand the ligand binding behavior of SDC4. In this study, expression and purification were performed to reveal structures of the short ecto-domain, the transmembrane domain, and the cytoplasmic domain of Syd4-eTC (SDC4). Solution-state NMR spectroscopy and solid-state NMR spectroscopy were used to study the structure of Syd4-eTC in membrane environments and to demonstrate the interaction between Syd4-eTC and PIP2.

Abstract 3725: ABI1 regulates STAT3 transcription through a DNA binding activity

Abstract Prostate cancer (PCa) is characterized by the complexity of oncogenic signaling and heterogeneity of transcriptional landscapes. Adaptor protein ABI1 is a tumor suppressor in PCa, as evidenced by its loss or downregulation in high-grade and metastatic tumors. STAT3 activation is a hallmark of high-risk prostate tumors. ABI1 loss is associated with STAT3 activation leading to transcriptional reprogramming and epithelial-mesenchymal-transition (EMT) of prostate cancer cells (Nath, Li et al. Cell Commun Signal. 2019). EMT changes involve several homeobox transcription factors. The fact that ABI1 contains a homeobox homology region (HHR) suggests the possibility that it plays a role in EMT by directly regulating transcriptional activity by DNA binding. To examine this hypothesis, we set out to analyze ABI1-DNA binding. Structural NMR studies and in-vitro protein-DNA binding assays confirmed ABI1 binding to DNA regulated by alternative spliced ABI1 Exon 4 region located on the C-terminus of ABI1 HHR. RNA-sequencing data and PCa cell lines qPCR assays reveal the alternative ABI1 Exon 4 spliced-in is enriched in high Gleason-scored PCa samples. ABI1 and STAT3 co-regulate their nuclear vs. cytoplasm localization, and the subsequent functional studies using PCa lines expressing wild type or HHR Exon 4 deletion mutant of ABI1 demonstrated that HHR regulates the STAT3 DNA binding patterns, STAT3 mediated chromatin structure programming as well as its transcription activities. We propose that ABI1 is a critical regulator of STAT3 activity during prostate cancer progression. Citation Format: Xiang Li, Neeru Arya, Baylee A. Porter, Allysa P. Kemraj, Xuesen Dong, Dominique Frueh, Alaji Bah, Leszek Kotula. ABI1 regulates STAT3 transcription through a DNA binding activity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3725.

1-ethyl-3-methyl imidazolium acetate, hemicellulolytic enzymes and laccase-mediator system: Toward an integrated co-valorization of polysaccharides and lignin from Miscanthus

The pretreatment of Miscanthus x giganteus with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]), followed by enzymatic hydrolysis and oxidative depolymerization, resulted in the efficient production of monomeric sugars and selective production of phenolic intermediates. The liquid fractions containing monomeric sugars were obtained with high yields (>80%), while a solid fraction enriched with lignin (>50% w.w−1) was isolated. The phenolic monomers released during [Emim][OAc]-pretreatment were identified and quantified by HPLC. A decrease in the content of vanillin, vanillic acid, and p-hydroxybenzaldehyde was observed during pretreatment, suggesting a selective solubilization of guaicyl and p-hydroxyphenyl units from the lignin polymer in [Emim][OAc]. The decrease in p-coumaric and ferulic acids content suggested a cleavage of lignin-carbohydrate complexes during pretreatment. Both pretreatment and enzymatic hydrolysis affected the content of ferulic acid ether and ester bonds. The NMR structural study (2D HSQC) of the isolated lignin showed the preservation of β-O-4′ interunit bonds, while 31P NMR analysis revealed a high content of aliphatic hydroxyl groups. The oxidative depolymerization of the isolated lignin, catalyzed by the laccase mediator system in the presence of 5% v.v−1 recycled [Emim][OAc] in buffer, mainly led to the production of free p-hydroxybenzaldehyde as a promising platform molecule. The mass balance of glucan, xylan and lignin from Miscanthus was validated throughout this strategy.

Orexin A, an amphipathic α-helical neuropeptide involved in pleiotropic functions in the nervous and immune systems: Synthetic approach and biophysical studies of the membrane-bound state

This research reports on the membrane interactions of orexin A (OXA), an α-helical and amphipathic neuropeptide that contains 33 residues and two disulfide bonds in the N-terminal region. OXA, which activates the orexins 1 and 2 receptors in neural and immune cell membranes, has essential pleiotropic physiological effects, including at the levels of arousal, sleep/wakefulness, energy balance, neuroprotection, lipid signaling, the inflammatory response, and pain. As a result, the orexin system has become a prominent target to treat diseases such as sleep disorders, drug addiction, and inflammation. While the high-resolution structure of OXA has been investigated in water and bound to micelles, there is a lack of information about its conformation bound to phospholipid membranes and its receptors. NMR is a powerful method to investigate peptide structures in a membrane environment. To facilitate the NMR structural studies of OXA exposed to membranes, we present a novel synthetic scheme, leading to the production of isotopically-labeled material at high purity. A receptor activation assay shows that the 15N-labeled peptide is biologically active. Biophysical studies are performed using surface plasmon resonance, circular dichroism, and NMR to investigate the interactions of OXA with phospholipid bilayers. The results demonstrate a strong interaction between the peptide and phospholipids, an increase in α-helical content upon membrane binding, and an in-plane orientation of the C-terminal region critical to function. This new knowledge about structure-activity relationships in OXA could inspire the design of novel therapeutics that leverage the anti-inflammatory and neuro-protective functions of OXA, and therefore could help address neuroinflammation, a major issue associated with neurological disorders such as Alzheimer's disease.

Identification of specific carbonic anhydrase inhibitors via in situ click chemistry, phage-display and synthetic peptide libraries: comparison of the methods and structural study.

The development of highly active and selective enzyme inhibitors is one of the priorities of medicinal chemistry. Typically, various high-throughput screening methods are used to find lead compounds from a large pool of synthetic compounds, and these are further elaborated and structurally refined to achieve the desired properties. In an effort to streamline this complex and laborious process, new selection strategies based on different principles have recently emerged as an alternative. Herein, we compare three such selection strategies with the aim of identifying potent and selective inhibitors of human carbonic anhydrase II. All three approaches, in situ click chemistry, phage-display libraries and synthetic peptide libraries, led to the identification of more potent inhibitors when compared to the parent compounds. In addition, one of the inhibitor-peptide conjugates identified from the phage libraries showed greater than 100-fold selectivity for the enzyme isoform used for the compound selection. In an effort to rationalize the binding properties of the conjugates, we performed detailed crystallographic and NMR structural analysis, which revealed the structural basis of the compound affinity towards the enzyme and led to the identification of a novel exosite that could be utilized in the development of isoform specific inhibitors.

High-affinity binding at quadruplex-duplex junctions: rather the rule than the exception.

Quadruplex-duplex (Q-D) junctions constitute unique structural motifs in genomic sequences. Through comprehensive calorimetric as well as high-resolution NMR structural studies, Q-D junctions with a hairpin-type snapback loop coaxially stacked onto an outer G-tetrad were identified to be most effective binding sites for various polycyclic quadruplex ligands. The Q-D interface is readily recognized by intercalation of the ligand aromatic core structure between G-tetrad and the neighboring base pair. Based on the thermodynamic and structural data, guidelines for the design of ligands with enhanced selectivity towards a Q-D interface emerge. Whereas intercalation at Q-D junctions mostly outcompete stacking at the quadruplex free outer tetrad or intercalation between duplex base pairs to varying degrees, ligand side chains considerably contribute to the selectivity for a Q-D target over other binding sites. In contrast to common perceptions, an appended side chain that additionally interacts within the duplex minor groove may confer only poor selectivity. Rather, the Q-D selectivity is suggested to benefit from an extension of the side chain towards the exposed part of the G-tetrad at the junction. The presented results will support the design of selective high-affinity binding ligands for targeting Q-D interfaces in medicinal but also technological applications.

4′‐SCF3‐Labeling Constitutes a Sensitive 19F NMR Probe for Characterization of Interactions in the Minor Groove of DNA

AbstractFluorinated nucleotides are invaluable for 19F NMR studies of nucleic acid structure and function. Here, we synthesized 4′‐SCF3‐thymidine (T ) and incorporated it into DNA by means of solid‐phase DNA synthesis. NMR studies showed that the 4′‐SCF3 group exhibited a flexible orientation in the minor groove of DNA duplexes and was well accommodated by various higher order DNA structures. The three magnetically equivalent fluorine atoms in 4′‐SCF3‐DNA constitute an isolated spin system, offering high 19F NMR sensitivity and excellent resolution of the positioning of T within various secondary and tertiary DNA structures. The high structural adaptability and high sensitivity of T make it a valuable 19F NMR probe for quantitatively distinguishing diverse DNA structures with single‐nucleotide resolution and for monitoring the dynamics of interactions in the minor groove of double‐stranded DNA.

4'-SCF3 -Labeling Constitutes a Sensitive 19 F NMR Probe for Characterization of Interactions in the Minor Groove of DNA.

Fluorinated nucleotides are invaluable for 19 F NMR studies of nucleic acid structure and function. Here, we synthesized 4'-SCF3 -thymidine (T ) and incorporated it into DNA by means of solid-phase DNA synthesis. NMR studies showed that the 4'-SCF3 group exhibited a flexible orientation in the minor groove of DNA duplexes and was well accommodated by various higher order DNA structures. The three magnetically equivalent fluorine atoms in 4'-SCF3 -DNA constitute an isolated spin system, offering high 19 F NMR sensitivity and excellent resolution of the positioning of T within various secondary and tertiary DNA structures. The high structural adaptability and high sensitivity of T make it a valuable 19 F NMR probe for quantitatively distinguishing diverse DNA structures with single-nucleotide resolution and for monitoring the dynamics of interactions in the minor groove of double-stranded DNA.

Ostrinia furnacalis PBP2 solution NMR structure: Insight into ligand binding and release mechanisms.

Ostrinia furnacalis is an invasive lepidopteran agricultural pest that relies on olfaction for mating and reproduction. Male moths have an extremely sensitive olfactory system that can detect the sex pheromones emitted by females over a great distance. Pheromone-binding proteins present in the male moth antenna play a key role in the pheromone uptake, transport, and release at the dendritic membrane of the olfactory neuron. Here, we report the first high-resolution NMR structure of a pheromone-binding protein from an Ostrinia species at pH 6.5. The core of the Ostrinia furnacalis PBP2 (OfurPBP2) consists of six helices, α1a (2-14), α1b (16-22), α2 (27-37), α3 (46-60), α4 (70-80), α5 (84-100), and α6 (107-124) surrounding a large hydrophobic pocket. The structure is stabilized by three disulfide bridges, 19-54, 50-108, and 97-117. In contrast to the unstructured C-terminus of other lepidopteran PBPs, the C-terminus of OfurPBP2 folds into an α-helix (α7) at pH 6.5. The protein has nanomolar affinity towards both pheromone isomers. Molecular docking of both pheromones, E-12 and Z-12-tetradecenyl acetate, to OfurPBP2 revealed that the residues Met5, Lys6, Met8, Thr9, Phe12, Phe36, Trp37, Phe76, Ser115, Phe118, Lys119, Ile122, His123, and Ala128 interact with both isomers, while Thr9 formed a hydrogen bond with the acetate head group. NMR structure and thermal unfolding studies with CD suggest that ligand release at pH 4.5 is likely due to the partial unfolding of the protein.

Abstract 2364: ABI1 regulates transcriptional activity of STAT3

Abstract Background: Prostate cancer (PCa) is characterized by the complexity of oncogenic signaling and heterogeneity of transcriptional landscapes. Adaptor protein ABI1 is a tumor suppressor in PCa as evidenced by its loss or downregulation in high grade and metastatic tumors. STAT3 activation is a hallmark of high-risk prostate tumors. ABI1 loss is associated with STAT3 activation leading to transcriptional reprogramming and epithelial-mesenchymal-transition (EMT) of prostate cancer cells (Nath, Li et al. 2019 Cell Commun Signal). EMT changes involve several homeobox transcription factors. The fact that ABI1 contains a homeobox homology region (HHR) suggested the possibility that it plays a role in EMT through directly regulating transcriptional activity by DNA binding. Methods: To examine this hypothesis we set out to analyze ABI1-DNA binding. We purified ABI1 HHR and demonstrated its in vitro interactions with different homeobox DNA-binding consensus of double-stranded DNA sequences. We created DU145 ABI1 CRISPR KO cell line and rescue ABI1 expression using retroviral transfection with either ABI1 wild type or Abi1 HHR mutants. DU145 cell lines STAT3 cellular localization was studied by Immunofluorescence staining and the STAT3 transcription activities was studied by Firefly Luciferase Assays with STAT3 specific luciferase reporter plasmid. Results: We found that the presence of alternatively spliced Exon 4-encoded sequence, located in the C-terminus of HHR, regulates binding of ABI1 to DNA. Structural NMR studies confirmed ABI1 binding to DNA. Subsequent functional studies using DU145 CRISPR KO cell lines expressing wild type or HHR mutants of ABI1 demonstrated that HHR regulates the nuclear localization and transcriptional activity of STAT3. Conclusion: We propose that ABI1 is a critical regulator of STAT3 activity during prostate cancer progression by the ABI1 HHR mediated DNA binding mechanism. Funding resources: NCI R01 CA161018; R21 CA260381 Conflicts of Interest Disclosure Statement: The authors declare no conflicts of interest. Citation Format: Xiang Li, Baylee Porter, Allysa Kemraj, Alaji Bah, Marcin Kortylewski, Leszek Kotula. ABI1 regulates transcriptional activity of STAT3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2364.

Clarifying the Use of Benzylidene Protecting Group for D-(+)-Ribono-1,4-Lactone, an Essential Building Block in the Synthesis of C-Nucleosides.

In the last two years, nucleosides analogues, a class of well-established bioactive compounds, have been the subject of renewed interest from the scientific community thanks to their antiviral activity. The COVID-19 global pandemic, indeed, spread light on the antiviral drug Remdesivir, an adenine C-nucleoside analogue. This new attention of the medical community on Remdesivir prompts the medicinal chemists to investigate once again C-nucleosides. One of the essential building blocks to synthetize these compounds is the D-(+)-ribono-1,4-lactone, but some mechanistic aspects linked to the use of different carbohydrate protecting groups remain unclear. Here, we present our investigations on the use of benzylidene as a ribonolactone protecting group useful in the synthesis of C-purine nucleosides analogues. A detailed 1D and 2D NMR structural study of the obtained compounds under different reaction conditions is presented. In addition, a molecular modeling study at the B3LYP/6-31G* level of theory with the SM8 solvation model for CHCl3 and DMSO to support the obtained results is used. This study allows for clarifying mechanistic aspects as the side reactions and structural rearrangements liked to the use of the benzylidene protecting group.

Emerging Contributions of Solid-State NMR Spectroscopy to Chromatin Structural Biology.

The eukaryotic genome is packaged into chromatin, a polymer of DNA and histone proteins that regulates gene expression and the spatial organization of nuclear content. The repetitive character of chromatin is diversified into rich layers of complexity that encompass DNA sequence, histone variants and post-translational modifications. Subtle molecular changes in these variables can often lead to global chromatin rearrangements that dictate entire gene programs with far reaching implications for development and disease. Decades of structural biology advances have revealed the complex relationship between chromatin structure, dynamics, interactions, and gene expression. Here, we focus on the emerging contributions of magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS NMR), a relative newcomer on the chromatin structural biology stage. Unique among structural biology techniques, MAS NMR is ideally suited to provide atomic level information regarding both the rigid and dynamic components of this complex and heterogenous biological polymer. In this review, we highlight the advantages MAS NMR can offer to chromatin structural biologists, discuss sample preparation strategies for structural analysis, summarize recent MAS NMR studies of chromatin structure and dynamics, and close by discussing how MAS NMR can be combined with state-of-the-art chemical biology tools to reconstitute and dissect complex chromatin environments.

Isotope-Labeled RNA Building Blocks for NMR Structure and Dynamics Studies.

RNA structural research lags behind that of proteins, preventing a robust understanding of RNA functions. NMR spectroscopy is an apt technique for probing the structures and dynamics of RNA molecules in solution at atomic resolution. Still, RNA analysis by NMR suffers from spectral overlap and line broadening, both of which worsen for larger RNAs. Incorporation of stable isotope labels into RNA has provided several solutions to these challenges. In this review, we summarize the benefits and limitations of various methods used to obtain isotope-labeled RNA building blocks and how they are used to prepare isotope-labeled RNA for NMR structure and dynamics studies.

C-kit2 G-quadruplex stabilized via a covalent probe: exploring G-quartet asymmetry.

Several sequences forming G-quadruplex are highly conserved in regulatory regions of genomes of different organisms and affect various biological processes like gene expression. Diverse G-quadruplex properties can be modulated via their interaction with small polyaromatic molecules such as pyrene. To investigate how pyrene interacts with G-rich DNAs, we incorporated deoxyuridine nucleotide(s) with a covalently attached pyrene moiety (Upy) into a model system that forms parallel G-quadruplex structures. We individually substituted terminal positions and positions in the pentaloop of the c-kit2 sequence originating from the KIT proto-oncogene with Upy and performed a detailed NMR structural study accompanied with molecular dynamic simulations. Our results showed that incorporation into the pentaloop leads to structural polymorphism and in some cases also thermal destabilization. In contrast, terminal positions were found to cause a substantial thermodynamic stabilization while preserving topology of the parent c-kit2 G-quadruplex. Thermodynamic stabilization results from π–π stacking between the polyaromatic core of the pyrene moiety and guanine nucleotides of outer G-quartets. Thanks to the prevalent overall conformation, our structures mimic the G-quadruplex found in human KIT proto-oncogene and could potentially have antiproliferative effects on cancer cells.

NMR structural study on the self-trimerization of d(GTTAGG) into a dynamic trimolecular G-quadruplex assembly preferentially in Na+ solution with a moderate K+ tolerance.

Vast G-quadruplexes (GQs) are primarily folded by one, two, or four G-rich oligomers, rarely with an exception. Here, we present the first NMR solution structure of a trimolecular GQ (tri-GQ) that is solely assembled by the self-trimerization of d(GTTAGG), preferentially in Na+ solution tolerant to an equal amount of K+ cation. Eight guanines from three asymmetrically folded strands of d(GTTAGG) are organized into a two-tetrad core, which features a broken G-column and two width-irregular grooves. Fast strand exchanges on a timescale of second at 17°C spontaneously occur between folded tri-GQ and unfolded single-strand of d(GTTAGG) that both species coexist in dynamic equilibrium. Thus, this tri-GQ is not just simply a static assembly but rather a dynamic assembly. Moreover, another minor tetra-GQ that has putatively tetrameric (2+2) antiparallel topology becomes noticeable only at an extremely high strand concentration above 18 mM. The major tri-GQ and minor tetra-GQ are considered to be mutually related, and their reversible interconversion pathways are proposed accordingly. The sequence d(GTTAGG) could be regarded as either a reading frame shifted single repeat of human telomeric DNA or a 1.5 repeat of Bombyx mori telomeric DNA. Overall, our findings provide new insight into GQs and expect more functional applications.

NMR study of magnetic structure and hyperfine interactions in the binary helimagnet FeP

We report a detailed study of the ground state helical magnetic structure in monophosphide FeP by means of ${}^{31}$P NMR spectroscopy. We show that the zero-field NMR spectrum of the polycrystalline sample provides strong evidence of an anisotropic distribution of local magnetic fields at the P site with substantially lower anharmonicity than that found at the Fe site by M\"{o}ssbauer spectroscopy. From field-sweep ${}^{31}$P NMR spectra we conclude that a continuous spin-reorientation transition occurs in an external magnetic field range of 4 - 7 T, which is also confirmed by specific-heat measurements. We observe two pairs of magnetically inequivalent phosphorus positions resulting in a pronounced four-peak structure of the single crystal ${}^{31}$P NMR spectra characteristic of an incommensurate helimagnetic ground state. We revealed a spatial redistribution of local fields at the P sites caused by Fe spin-reorientation transition in high fields and developed an effective approach to account for it. We demonstrate that all observed ${}^{31}$P spectra can be treated within a model of an isotropic helix of Fe magnetic moments in the ($ab$)-plane with a phase shift of 36$^{\circ}$ and 176$^{\circ}$ between Fe1-Fe3 (Fe2-Fe4) and Fe1-Fe2 (Fe3-Fe4) sites, respectively, in accordance with the neutron scattering data.

Structure of a DNA G-Quadruplex Related to Osteoporosis with a G-A Bulge Forming a Pseudo-loop.

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).