NMR Structural Studies Research Articles

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).

NMR Structure and Dynamics Studies of Yeast Respiratory Supercomplex Factor 2

The Saccharomyces cerevisiae respiratory supercomplex factor 2 (Rcf2) is a 224-residue protein located in the mitochondrial inner membrane where it is involved in the formation of supercomplexes composed of cytochrome bc1 and cytochrome c oxidase. We previously demonstrated that Rcf2 forms a dimer in dodecylphosphocholine micelles, and here we report the solution NMR structure of this Rcf2 dimer. Each Rcf2 monomer has two soluble α helices and five putative transmembrane (TM) α helices, including an unexpectedly charged TM helix at the C terminus, which mediates dimer formation. The NOE contacts indicate the presence of inter-monomer salt bridges and hydrogen bonds at the dimer interface, which stabilize the Rcf2 dimer structure. Moreover, NMR chemical shift change mapping upon lipid titrations as well as molecular dynamics analysis reveal possible structural changes upon embedding Rcf2 into a native lipid environment. Our results contribute to the understanding of respiratory supercomplex formation and regulation.

NMR structural studies and mechanism of action of Lactophoricin analogs as antimicrobial peptides

Antimicrobial peptides (AMPs) are effective alternatives to conventional antibiotics. They protect the host from the constant invasion of a broad range of infectious microorganisms. AMPs have been at the forefront of the response to multidrug-resistant microbial strains and appear to be ideal drug candidates. Lactophoricin (LPcin), naturally produced from bovine milk, is a typical cationic antimicrobial peptide. Three analog peptides, including LPcin-YK5, LPcin-YK8, and LPcin-YK11, with enhanced antimicrobial activity compared to the wild-type LPcin, were designed and expressed in our laboratory. We investigated the structure and antimicrobial mechanisms of action of the three novel antimicrobial peptide analogs derived from LPcin using solution NMR and solid-state NMR spectroscopy in membrane environments. Our results revealed that the three LPcin analogs exhibited helical structures with different tilt angles on the phospholipid membrane surface. We proposed three-dimensional conformations and antibacterial mechanisms of action of the three peptide analogs in the phospholipid bilayers using two-dimensional solid-state separated local field NMR experiments.

Structural features of the bioactive cyanobacterium Nostoc sp. exopolysaccharide

Microalgal biopolymers are studied mainly in terms of physico-chemical characterization, biological effects as well as possible biotechnological applications. Due to the significant antitussive, bronchodilator, anti-inflammatory and immunomodulatory effects of the previously isolated crude extracellular polysaccharide (EPS) produced by the cyanobacterium Nostoc sp., the purified biopolymer and its oligosaccharides, obtained after partial acid hydrolysis, were subjected to an in-depth NMR structural study. Analyses of the data obtained by chemical methods and NMR showed that the EPS backbone is composed of the repeating unit [→4)-β-D-Xylp-(1 → 4)-β-D-Glcp-(1 → 4)-α-L-Arap-(1 → 3)-β-D-Manp-(1→]n, in which about 60% of glucose units are substituted at C6 by uronic acids, in particular by the unusual unsaturated 3-O-lactyl-4-deoxy-α-erythro-hex-4-enopyranuronic acid, and to a lesser extent by β-D-glucuronic acid and 3-O-lactyl-β-D-glucuronic acid. These findings, structural features and identified biological effects, suggest the potential use of this biopolymer in the medical-pharmaceutical field.

Synthesis of paramagnetic ligands that target the C-terminal binding site of Hsp90

Identification of a ligand binding site represents the starting point for a structure-based drug development program. Lack of a binding site hampers the development of improved ligands that modulate the protein of interest. In this letter, we describe the development of chemical tools that will allow for elucidation of the Hsp90 C-terminal ligand binding site. Our strategy is based on the preparation of paramagnetic analogs of KU-596, an investigational new drug that is currently undergoing clinical trials for the treatment of neuropathy and interacts with the Hsp90 C-terminal domain. In particular, we report the design and synthesis of three novel paramagnetic analogs of KU-596, which will be used to obtain long range distances for NMR structural studies of Hsp90 in complex with C-terminal ligands.

Effect of Phosphorylation on the Structural Behaviour of Peptides Derived from the Intrinsically Disordered C-Terminal Domain of Histone H1.0.

To investigate the structural impact of phosphorylation on the human histone H1.0 C-terminal domain, we performed NMR structural studies of model peptides containing a single phosphorylation site: T118 -H1.0 (T118 PKK motif) and T140 -H1.0 (T140 PVK motif). Both model peptides are mainly disordered in aqueous solution in their non-phosphorylated and phosphorylated forms, but become structured in the presence of trifluoroethanol. The peptides T118 -H1.0 and pT118 -H1.0 contain two helical regions, a long amphipathic α helix spanning residues 104-115 and a short α/310 helix (residues 119-123), that are almost perpendicular in T118 -H1.0 but have a poorly defined orientation in pT118 -H1.0. Peptides T140 -H1.0 and pT140 -H1.0 form very similar α helices between residues 141-147. The TPKK and TPVK motifs show the same backbone conformation, but differ in their side-chain contacts; the Thr and pThr side chains interact with the i+2 Lys side chain in the TPKK motif, and with the i+3 Lys side chain in the TPVK motif. The pT phosphate group in pT118 -H1.0 and pT140 -H1.0 has pKa values below the intrinsic values, which can be explained by non-specific charge-charge interactions with nearby Lys. The non-polar Val in the TPVK motif accounts for the pT140 pKa being closer to the intrinsic pKa value than the pT118 pKa . Altogether, these results validate that minimalist strategies using model peptides can provide structural details difficult to obtain in short-lived intrinsically disordered proteins and domains.

Larp6 Regulates Collagen mRNA by Targeted Unwinding of a Conserved Stem‐Loop

Collagen is the most abundant protein in the human body and a key component of the extracellular matrix. Regulation of collagen production in key to the development of fibroproliferative disorders; everything from scarring to cardiovascular disease. La ribonucleoprotein 6 (Larp6) is an RNA binding protein which has recently been shown to regulate collagen production by binding to a conserved region at the translation start site of collagen mRNA. This interaction appears to be critical to directing RNA to the rough ER for translation initiation. Additionally, LARP6 interaction with a number of molecular chaperones appears to be important for regulating collagen production. We hypothesize that LARP6 utilizes conformational selection in the recognition and discrimination of collagen mRNAs and that tertiary interactions dictate mRNA fate. In vitro RNA chaperone assays, translation assays, RNAi, confocal microscopy, and NMR and SAX structural studies are employed to interrogate these interactions. New information regarding the structures and dynamics that determine the Larp6 interaction with collagen mRNA may inform novel therapeutic targets that can mitigate fibroproliferative disease.Support or Funding InformationP20GM109095

Mutation of Conserved Mre11 Residues Alter Protein Dynamics to Separate Nuclease Functions

Naked and protein-blocked DNA ends occur naturally during immune cell development, meiosis, and at telomeres as well as from aborted topoisomerase reactions, collapsed replication forks, and other stressors. Damaged DNA ends are dangerous in cells and if left unrepaired can lead to genomic rearrangement, loss of genetic information, and eventually cancer. Mre11 is part of the Mre11–Rad50–Nbs1 complex that recognizes DNA double-strand breaks and has exonuclease and endonuclease activities that help to initiate the repair processes to resolve these broken DNA ends. In fact, these activities are crucial for proper DNA damage repair pathway choice. Here, using Pyrococcus furiosus Mre11, we question how two Mre11 separation-of-function mutants, one previously described but the second first described here, maintain endonuclease activity in the absence of exonuclease activity. To start, we performed solution-state NMR experiments to assign the side-chain methyl groups of the 64-kDa Mre11 nuclease and capping domains, which allowed us to describe the structural differences between Mre11 bound to exo- and endonuclease substrates. Then, through biochemical and biophysical characterization, including NMR structural and dynamics studies, we compared the two mutants and determined that both affect the dynamic features and double-stranded DNA binding properties of Mre11, but in different ways. In total, our results illuminate the structural and dynamic landscape of Mre11 nuclease function.

Impact of a Snap-Back Loop on Stability and Ligand Binding to a Parallel G-Quadruplex.

Various genomic DNA sequences including a MYC promoter sequence are amenable to the formation of a G-quadruplex featuring a snap-back loop with the incorporation of a 3'-terminal guanine into the quadruplex core. To evaluate relative stabilities and ligand binding in more detail, optical, microcalorimetric, and NMR structural studies were performed on both a minimal mutant sequence Pu22T that exclusively folds into a snap-back loop quadruplex and a parallel MYC quadruplex proposed to be the most relevant fold of the MYC promoter in a cellular environment. Similar thermal stabilities for Pu22T and MYC suggest the coexistence of both quadruplexes when derived from a sequence able to fold into both topologies. Isothermal titration calorimetry indicates a mostly identical enthalpy-driven strong binding of an indoloquinoline ligand but with a reduced number of high-affinity binding sites in Pu22T in line with a novel modified FRET competitive melting assay. Corroborated by fluorescence titrations using 2-aminopurine as a fluorescent probe, NMR chemical shift footprints show binding of the ligand at the Pu22T 5'-outer tetrad with the formation of a binding pocket. On the other hand, steric restrictions due to the snap-back loop severely restrict ligand stacking on the 3'-outer tetrad of Pu22T.

NMR and crystallographic structural studies of the Elongation factor P from Staphylococcus aureus.

Elongation factor P (EF-P) is a translation protein factor that plays an important role in specialized translation of consecutive proline amino acid motifs. EF-P is an essential protein for cell fitness in native environmental conditions. It regulates synthesis of proteins involved in bacterial motility, environmental adaptation and bacterial virulence, thus making EF-P a potential drug target. In the present study, we determined the solution and crystal structure of EF-P from the pathogenic bacteria Staphylococcus aureus at 1.48Å resolution. The structure can serve as a platform for structure-based drug design of novel antibiotics to combat the growing antibiotic resistance of S. aureus.

Characterization of the Human KCNQ1 Voltage Sensing Domain (VSD) in Lipodisq Nanoparticles for Electron Paramagnetic Resonance (EPR) Spectroscopic Studies of Membrane Proteins.

Membrane proteins are responsible for conducting essential biological functions that are necessary for the survival of living organisms. In spite of their physiological importance, limited structural information is currently available as a result of challenges in applying biophysical techniques for studying these protein systems. Electron paramagnetic resonance (EPR) spectroscopy is a very powerful technique to study the structural and dynamic properties of membrane proteins. However, the application of EPR spectroscopy to membrane proteins in a native membrane-bound state is extremely challenging due to the complexity observed in inhomogeneity sample preparation and the dynamic motion of the spin label. Detergent micelles are very popular membrane mimetics for membrane proteins due to their smaller size and homogeneity, providing high-resolution structure analysis by solution NMR spectroscopy. However, it is important to test whether the protein structure in a micelle environment is the same as that of its membrane-bound state. Lipodisq nanoparticles or styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) have been introduced as a potentially good membrane-mimetic system for structural studies of membrane proteins. Recently, we reported on the EPR characterization of the KCNE1 membrane protein having a single transmembrane incorporated into lipodisq nanoparticles. In this work, lipodisq nanoparticles were used as a membrane mimic system for probing the structural and dynamic properties of the more complicated membrane protein system human KCNQ1 voltage sensing domain (Q1-VSD) having four transmembrane helices using site-directed spin-labeling EPR spectroscopy. Characterization of spin-labeled Q1-VSD incorporated into lipodisq nanoparticles was carried out using CW-EPR spectral line shape analysis and pulsed EPR double-electron electron resonance (DEER) measurements. The CW-EPR spectra indicate an increase in spectral line broadening with the addition of the styrene-maleic acid (SMA) polymer which approaches close to the rigid limit providing a homogeneous stabilization of the protein-lipid complex. Similarly, EPR DEER measurements indicated a superior quality of distance measurement with an increase in the phase memory time (Tm) values upon incorporation of the sample into lipodisq nanoparticles when compared to proteoliposomes. These results are consistent with the solution NMR structural studies on the Q1-VSD. This study will be beneficial for researchers working on investigating the structural and dynamic properties of more complicated membrane protein systems using lipodisq nanoparticles.

Synthesis, complete NMR assignment and structural study of a steroidal dimer of 17α-ethynyl-5α,10α-estran-17β-ol with diethynylbenzene spacer

A phenylene-bridged steroidal dimer derived from 17α-ethynyl-5α,10α-estran-17β-ol with molecular rotor-like architecture was synthesized to investigate the supramolecular interactions directing the crystallization of these systems. Structures with varying importance in complementarity between H-bonding and hydrophobic interactions can be observed directing the packing of the obtained crystals, depending on the synthetic stage, though conserving the same space group for both systems. Such behavior clearly shows the versatility achievable using steroids as crystal packing directors. Alongside this structural study, the complete NMR assignment is presented for the dimer, and precursors, in which the steroids present an unconventional and noteworthy A-B ring fusion.

NMR Structural Studies and Antibacterial Killing Mechanisms of Antimicrobial Peptides with Higher Activity

NMR Structural Studies and Antibacterial Killing Mechanisms of Antimicrobial Peptides with Higher Activity

Intercalation of a Heterocyclic Ligand between Quartets in a G-Rich Tetrahelical Structure.

YES G‐rich oligonucleotide VK2 folds into an AGCGA‐quadruplex tetrahelical structure distinct and significantly different from G‐quadruplexes, even though it contains four G3 tracts. Herein, a bis‐quinolinium ligand 360A with high affinity for G‐quadruplex structures and selective telomerase inhibition is shown to strongly bind to VK2. Upon binding, 360A does not induce a conformational switch from VK2 to an expected G‐quadruplex. In contrast, NMR structural study revealed formation of a well‐defined VK2–360A complex with a 1:1 binding stoichiometry, in which 360A intercalates between GAGA‐ and GCGC‐quartets in the central cavity of VK2. This is the first high‐resolution structure of a G‐quadruplex ligand intercalating into a G‐rich tetrahelical fold. This unique mode of ligand binding into tetrahelical DNA architecture offers insights into the stabilization of an AGCGA‐quadruplex by a heterocyclic ligand and provides guidelines for rational design of novel VK2 binding molecules with selectivity for different DNA secondary structures.

Biological and chemical insight into Gaultheria procumbens fruits: a rich source of anti-inflammatory and antioxidant salicylate glycosides and procyanidins for food and functional application.

The fruits of Gaultheria procumbens are traditionally used for culinary and healing purposes as anti-inflammatory agents. In the present work, the active components of the fruits were identified (UHPLC-PDA-ESI-MS3, preparative HPLC isolation, and NMR structural studies), and their biological capacity was evaluated in vitro in cell-based and non-cellular models. The fruits were revealed to be the richest known dietary source of salicylates (38.5 mg per g fruit dw). They are also rich in procyanidins (28.5 mg per g fruit dw). Among five tested solvents, acetone was the most efficient in concentrating the phenolic matrix (39 identified compounds; 191.3 mg g-1, 121.7 mg g-1, and 50.9 mg g-1 dry extract for total phenolics, salicylates, and procyanidins, respectively). In comparison to positive controls (dexamethasone, indomethacin, and quercetin), the extract (AE) and pure salicylates exhibited strong inhibitory activity towards pro-inflammatory enzymes (cyclooxygenase-2 and hyaluronidase). The analytes were found to be non-cytotoxic (flow cytometry) towards human neutrophils ex vivo. Moreover, they significantly, in a dose-dependent manner, downregulated the release of ROS, TNF-α, IL-1β, and elastase-2 and slightly inhibited the secretion of IL-8 and metalloproteinase-9 in the cells. The observed effects might support the usage of G. procumbens fruits as functional components of an anti-inflammatory diet and indicate the potential of AE for use in adjuvant treatment of inflammatory disorders cross-linked with oxidative stress and associated with the excessive production of TNF-α, IL-1β, and elastase-2.

NMR Study of Spatial Structure and Internal Dynamic of Adducts of Ninhydrin-Derived Azomethine Ylide with Cyclopropenes

Using NMR spectroscopy, the structure of the new products obtained in reaction of ninhydrin-derived azomethine ylide with cyclopropenes was established and conformational mobility was studied. Complete signal assignments in 1H and 13C spectra on base analysis of some homo- and heteronuclear correlation spectra were made, and qualitative comparison of the cross-peak intensities in the NOESY spectrum confirmed the structure of compounds 4a–4c in which three aromatic rings “a”, “b” and “c” at carbons of the cyclopropane ring are facing to us and the side substitute group at carbon C10 is situated in back direction. The hindered rotations of these aromatic rings in rigid part of molecules under study were found and values of free energy activation ∆G≠ of these rotations were evaluated on base coalescence temperature (Tc) and frequency difference (∆ν) data. The existence of fast in NMR time-scale conformational equilibrium in the solution associated with hindered rotation of side substitute group around C10–C14 bond was proved. In case of adduct 4c, the ratio 68:32 of two preferable conformers has been determined at room temperature in CDCl3 on base a comparison of calculated and experimental values of vicinal constant 3J10α–14β. This scalar constant turned out to be 1.0 Hz more for adduct 4a and this reflects an increase in the population of the main conformer almost by 10%. In the NOESY spectra of all studied adducts, a very rare phenomenon of scalar relaxation was found. It manifests itself in appearance of the additional scalar input into the effective cross-relaxation rate between two protons, and in case ωoτc ≪ 1, it may lead to a change in the sign of the cross-peak to the opposite if the scalar constant between them JH–H turns out to be time-dependent (scalar relaxation of the first kind) or in case of quadrupole nuclear situated near from one of these protons (scalar relaxation of the second kind). Thus, the conformational mobility which is connected with fast (in NMR time scale) nitrogen inversion in five-membered ring of adducts under study was proved due to the presence of the so-called scalar relaxation effects in NOESY spectra which look as a change cross-peak sign in comparison with the usual case.

Solution NMR Structural Studies of Glycans

AbstractIn this account, we present a summary of our progress in the study of glycan solution conformations by NMR spectroscopy. We begin by comparing the structural biology of glycans to that of proteins. We continue by defining the challenges in solution structural studies of glycans as we see them and show what we have done to help address those challenges. This includes Residual Dipolar Coupling studies and their hurdles, accurate coupling constant measurement in the presence of strong 1H‐1H coupling, direct detection of hydrogen bonds where the NHs, OHs or CHs are the hydrogen bond donors, and on‐cell NMR structural studies. We also suggest some future approaches to glycan structure determination.

Metal‐Chelated Polymer Nanodiscs for NMR Studies

AbstractParamagnetic relaxation enhancement (PRE) is commonly used to speed up spin lattice relaxation time (T1) for rapid data acquisition in NMR structural studies. Consequently, there is significant interest in novel paramagnetic labels for enhanced NMR studies on biomolecules. Herein, we report the synthesis and characterization of a modified poly(styrene‐co‐maleic acid) polymer which forms nanodiscs while showing the ability to chelate metal ions. Cu2+‐chelated nanodiscs are demonstrated to reduce the T1 of protons for both polymer and lipid‐nanodisc components. The chelated nanodiscs also decrease the proton T1 values for a water‐soluble DNA G‐quadruplex. These results suggest that polymer nanodiscs functionalized with paramagnetic tags can be used to speed‐up data acquisition from lipid bilayer samples and also to provide structural information from water‐soluble biomolecules.

Metal-Chelated Polymer Nanodiscs for NMR Studies.

Paramagnetic relaxation enhancement (PRE) is commonly used to speed up spin lattice relaxation time (T1 ) for rapid data acquisition in NMR structural studies. Consequently, there is significant interest in novel paramagnetic labels for enhanced NMR studies on biomolecules. Herein, we report the synthesis and characterization of a modified poly(styrene-co-maleic acid) polymer which forms nanodiscs while showing the ability to chelate metal ions. Cu2+ -chelated nanodiscs are demonstrated to reduce the T1 of protons for both polymer and lipid-nanodisc components. The chelated nanodiscs also decrease the proton T1 values for a water-soluble DNA G-quadruplex. These results suggest that polymer nanodiscs functionalized with paramagnetic tags can be used to speed-up data acquisition from lipid bilayer samples and also to provide structural information from water-soluble biomolecules.

Fast MAS 1H-13C correlation NMR for structural investigations of plant cell walls.

Plant cell walls consist of a mixture of polysaccharides that render the cell wall a strong and dynamic material. Understanding the molecular structure and dynamics of wall polysaccharides is important for understanding and improving the properties of this energy-rich biomaterial. So far, solid-state NMR studies of cell wall structure and dynamics have solely relied on 13C chemical shifts measured from 2D and 3D correlation experiments. To increase the spectral resolution, sensitivity and upper limit of measurable distances, it is of interest to explore 1H chemical shifts and 1H-detected NMR experiments for analyzing cell walls. Here we demonstrate 2D and 3D 1H-13C correlation experiments at both moderate and fast MAS frequencies of 10-50kHz to resolve and assign 1H chemical shifts of matrix polysaccharides in Arabidopsis primary cell walls. Both 13C-detected and 1H-detected experiments are implemented and are shown to provide useful and complementary information. Using the assigned 1H chemical shifts, we measured long-range correlations between matrix polysaccharides and cellulose using 1H-1H instead of 13C-13C spin diffusion, and the 2D experiments can be conducted with either 13C or 1H detection.