A novel cGUUAg tetraloop structure with a conserved yYNMGg-type backbone conformation from cloverleaf 1 of bovine enterovirus 1 RNA

The 5′-terminal cloverleaf (CL)-like RNA structures are essential for the initiation of positive- and negative-strand RNA synthesis of entero- and rhinoviruses. SLD is the cognate RNA ligand of the viral proteinase 3C (3C(pro)), which is an indispensable component of the viral replication initiation complex. The structure of an 18mer RNA representing the apical stem and the cGUUAg D-loop of SLD from the first 5′-CL of BEV1 was determined in solution to a root-mean-square deviation (r.m.s.d.) (all heavy atoms) of 0.59 Å (PDB 1Z30). The first (antiG) and last (synA) nucleotide of the D-loop forms a novel ‘pseudo base pair’ without direct hydrogen bonds. The backbone conformation and the base-stacking pattern of the cGUUAg-loop, however, are highly similar to that of the coxsackieviral uCACGg D-loop (PDB 1RFR) and of the stable cUUCGg tetraloop (PDB 1F7Y) but surprisingly dissimilar to the structure of a cGUAAg stable tetraloop (PDB 1MSY), even though the cGUUAg BEV D-loop and the cGUAAg tetraloop differ by 1 nt only. Together with the presented binding data, these findings provide independent experimental evidence for our model [O. Ohlenschläger, J. Wöhnert, E. Bucci, S. Seitz, S. Häfner, R. Ramachandran, R. Zell and M. Görlach (2004) Structure, 12, 237–248] that the proteinase 3C(pro) recognizes structure rather than sequence

Structural insights into heme binding to IL-36α proinflammatory cytokine

Cytokines of the interleukin (IL)-1 family regulate immune and inflammatory responses. The recently discovered IL-36 family members are involved in psoriasis, rheumatoid arthritis, and pulmonary diseases. Here, we show that IL-36α interacts with heme thereby contributing to its regulation. Based on in-depth spectroscopic analyses, we describe two heme-binding sites in IL-36α that associate with heme in a pentacoordinated fashion. Solution NMR analysis reveals structural features of IL-36α and its complex with heme. Structural investigation of a truncated IL-36α supports the notion that the N-terminus is necessary for association with its cognate receptor. Consistent with our structural studies, IL-36-mediated signal transduction was negatively regulated by heme in synovial fibroblast-like synoviocytes from rheumatoid arthritis patients. Taken together, our results provide a structural framework for heme-binding proteins and add IL-1 cytokines to the group of potentially heme-regulated proteins

Solution structure of stem-loop α of the hepatitis B virus post-transcriptional regulatory element

Chronic hepatitis B virus (HBV) infections may lead to severe diseases like liver cirrhosis or hepatocellular carcinoma (HCC). The HBV post-transcriptional regulatory element (HPRE) facilitates the nuclear export of unspliced viral mRNAs, contains a splicing regulatory element and resides in the 3′-region of all viral transcripts. The HPRE consists of three sub-elements α (nucleotides 1151–1346), β1 (nucleotides 1347–1457) and β2 (nucleotides 1458–1582), which confer together full export competence. Here, we present the NMR solution structure (pdb 2JYM) of the stem-loop α (SLα, nucleotides 1292–1321) located in the sub-element α. The SLα contains a CAGGC pentaloop highly conserved in hepatoviruses, which essentially adopts a CUNG-like tetraloop conformation. Furthermore, the SLα harbours a single bulged G residue flanked by A-helical regions. The structure is highly suggestive of serving two functions in the context of export of unspliced viral RNA: binding sterile alpha motif (SAM-) domain containing proteins and/or preventing the utilization of a 3′-splice site contained within SLα

Zeeman perturbed spin echo FT-NQR spectroscopy

An approach for the evaluation of the asymmetry parameter, η, of the electric field gradient tensor for spin I = 3 2 systems from Zeeman NQR spectroscopy of polycrystalline samples is presented. The method relies on Fourier transformation of the second half of the Zeeman-perturbed Hahn spin-echo signal, generated via a two-pulse excitation sequence. Suitable phase cycling of the RF pulses of the spin-echo sequence has been derived and implemented, for the first time in NQR spectroscopy, to retain only the echo component in the detected signal. The RF pulse phase-cycling scheme employed in this work permits the extraction of the parameters of interest with improved sensitivity. A numerical procedure for simulating the powder Zeeman spin-echo FT NQR spectra is also given. A comparison of the theoretically simulated spin-echo spectrum with that recorded experimentally is shown to permit the evaluation of η. The method has been successfully demonstrated for the two 35Cl sites in 2,4,6-trichloro-1,3,5-triazine ("cyanuric chloride")

Measurement of the tensor-asymmetry parameter of the electric-field gradient for spin-3/2 nuclei in polycrystalline specimens via Zeeman FT NQR spectroscopy

A knowledge of vQ alone is insufficient to independently determine the two structural parameters of interest, namely the nuclear quadrupole coupling constant e*qQ and the electric-field-gradient tensor-asymmetry parameter 1. Different approaches, with varying degrees of utility, have been presented in the literature (2-12) for obtaining these parameters. In situations where a highly homogeneous RF field of appreciable strength is available over the entire sample volume, Harbison et al. ( 11, 12) have recently demonstrated the elucidation of 4 from an analysis of the two-dimensional zero-field nutation NQR spectral data. Alternatively, when the inhomogeneous NQR linewidth is not very large, a simpler one-dimensional approach for the extraction of 17 involves the application of a Zeeman perturbation to lift the degeneracy of the quadrupole energy levels and a subsequent analysis of the Zeeman spectra (2-9). The signal-to-noise ratios encountered in the Zeeman NQR investigations of polycrystalline materials are often very low and signal averaging is a prerequisite for obtaining highfidelity spectral lineshape. Because of the higher sensitivity and ease with which longterm signal averaging can be effected, transient rather than steady-state techniques are obviously the method of choice for 7 determination. However, in the analysis of the Zeeman FT NQR spectra, one must take into consideration factors such as the receiver dead time and the strength of the RF field in relation to the magnitude of the Zeeman field and the resonance offset

NMR solution structure of a parallel LNA quadruplex

The solution structure of a locked nucleic acid (LNA) quadruplex, formed by the oligomer d(TGGGT), containing only conformationally restricted LNA residues is reported. NMR and CD spectroscopy, as well as molecular dynamics and mechanic calculations, has been used to characterize the complex. The molecule adopts a parallel stranded conformation with a 4-fold rotational symmetry, showing a right-handed helicity and the guanine residues in an almost planar conformation with three well-defined G-tetrads. The thermal stability of Q-LNA has been found to be comparable with that of [r(UGGGU)](4), while a T(m) increment of 20°C with respect to the corresponding DNA quadruplex structure [d(TGGGT)](4) has been observed. The structural features of the LNA quadruplex reported here may open new perspectives for the biological application of LNAs as novel versatile tools to design aptamer or catalyst oligonucleotides

Structural insights into a wildtype domain of the oncoprotein E6 and its interaction with a PDZ domain.

The high-risk human papilloma virus (HPV) oncoproteins E6 and E7 interact with key cellular regulators and are etiological agents for tumorigenesis and tumor maintenance in cervical cancer and other malignant conditions. E6 induces degradation of the tumor suppressor p53, activates telomerase and deregulates cell polarity. Analysis of E6 derived from a number of high risk HPV finally yielded the first structure of a wild-type HPV E6 domain (PDB 2M3L) representing the second zinc-binding domain of HPV 51 E6 (termed 51Z2) determined by NMR spectroscopy. The 51Z2 structure provides clues about HPV-type specific structural differences between E6 proteins. The observed temperature sensitivity of the well-folded wild-type E6 domain implies a significant malleability of the oncoprotein in vivo. Hence, the structural differences between individual E6 and their malleability appear, together with HPV type-specific surface exposed side-chains, to provide the structural basis for the different interaction networks reported for individual E6 proteins. Furthermore, the interaction of 51Z2 with a PDZ domain of hDlg was analyzed. Human Dlg constitutes a prototypic representative of the large family of PDZ proteins regulating cell polarity, which are common targets of high-risk HPV E6. Nine C-terminal residues of 51Z2 interact with the second PDZ domain of hDlg2. Surface plasmon resonance in conjunction with the NMR spectroscopy derived complex structure (PDB 2M3M) indicate that E6 residues N-terminal to the canonical PDZ-BM of E6 significantly contribute to this interaction and increase affinity. The structure of the complex reveals how residues outside of the classical PDZ-BM enhance the affinity of E6 towards PDZ domains. Such mechanism facilitates successful competition of E6 with cellular PDZ-binding proteins and may apply to PDZ-binding proteins of other viruses as well

Three-dimensional structure of the S4-S5 segment of the Shaker potassium channel.

The propagation of action potentials during neuronal signal transduction in phospholipid membranes is mediated by ion channels, a diverse group of membrane proteins. The S4-S5 linker peptide (S4-S5), that connects the S4 and S5 transmembrane segments of voltage-gated potassium channels is an important region of the Shaker ion-channel protein. Despite its importance, very little is known about its structure. Here we provide evidence for an amphipathic alpha-helical conformation of a synthetic S4-S5 peptide of the voltage-gated Drosophila melanogaster Shaker potassium channel in water/trifluoroethanol and in aqueous phospholipid micelles. The three-dimensional solution structures of the S4-S5 peptide were obtained by high-resolution nuclear magnetic resonance spectroscopy and distance-geometry/simulated-annealing calculations. The detailed structural features are discussed with respect to model studies and available mutagenesis data on the mechanism and selectivity of the potassium channel

Virtual coupling effects in heteronuclear chemical-shift correlation spectroscopy

Heteronuclear chemical-shift correlation (HETCOR) spectroscopy ( I, 2) has become an important 2D NMR technique in the study of complex organic and bioorganic molecules. The cross peaks in the 2D spectra normally arise due to the existence of a nonvanishing heteronuclear scalar coupling. However, virtual coupling between ‘H and 13C spins (3- 7)) arising due to strong coupling between two or more protons, can result in the appearance of spurious peaks in the HETCOR spectra. The commonly used HETCOR pulse sequences are shown in Figs. 1 A and 1 B. Sequence 1 A provides 2D spectra with complete suppression of heteronuclear couplings in both dimensions, although ‘H- ‘H scalar couplings remain in wl. Sequence 1B with a BIRD sandwich (8) at the center of the evolution period provides improved resolution and sensitivity by virtue of its ability to suppress scalar interactions between all but the nonequivalent geminal protons. Complete ‘H- ‘H decoupling ( 9-12) in the HETCOR spectra can provide further improvements and sequence 1 C with its origin in the widely used COLOC sequence (13) is one of the simplest sequences that can provide such spectra with signal intensities dependent only on the homonuclear couplings. In situations involving a wide range of homonuclear ‘H- ‘H couplings sequence 1 C can be conveniently modified by replacing the 180” (C) pulse at the center of the constant time T by a BIRD sandwich, to remove the dependence of the crosspeak intensities on the homonuclear couplings

Structural Studies on LNA Quadruplexes

LNAs (locked nucleic acids) are new DNA analogs with higher binding affinities toward nucleic acids than the canonical counterparts mainly due to the characteristic conformational restriction arising from the 2'-O, 4'-C methylene bridge. In light of the promising therapeutic applications and considering the advantageous characteristics of LNAs, such as their high water soly., easy handling, and synthetic accessibility through the conventional phosphoramidite chem., we undertook a study concerning the capability of these nucleic acid analogs to form quadruplex structures. Particularly, we have been investigating the LNA/DNA chimeras corresponding to the well-known DNA sequences 5'-GGTTGGTGTGGTTGG-3', capable of forming an unimol. quadruplex. This article deals with the study of the sequence 5'-ggTTggTGTggTTgg-3' (upper and lower case letters represent DNA and LNA residues, resp.), which, according to CD spectroscopy, is able to fold into a quadruplex structure