No abstract

The solution structure of domain III from the New York West Nile virus strain 385-99 (WN-rED3) has been determined by NMR methods. The West Nile domain III structure is a ␤-barrel structure formed from seven antiparallel ␤-strands in two ␤-sheets. One anti-parallel ␤-sheet consists of ␤-strands ␤1 In 2002, the mosquito-borne West Nile virus (WNV) 1 (family Flaviviridae, genus Flavivirus) was responsible for the largest outbreak of arthropod-borne encephalitis recorded in the Western hemisphere. In that year, 4156 human infections and 284 deaths were reported in the United States (1). In 2003, the WNV epidemic continued with large numbers of human and animal disease across North America, and it was detected for the first time in Mexico and Central America (e.g. Refs. 2-4). The objective of this study was to solve the solution structure for the putative envelope (E) protein, the receptor-binding domain of WNV with the long term goal of using these findings for the development of structure-based vaccines or antiviral agents. Currently, there are no approved vaccines for WNV or therapeutic treatments for West Nile encephalitis.The flaviviruses are small, enveloped, positive-sense RNA viruses that are transmitted primarily by either mosquitoes or ticks. The translation of the single open reading frame within the viral genome, followed by co-and posttranslational cleavage, results in ten viral proteins, three structural proteins (core, membrane, and E) and seven non-structural proteins. The non-structural proteins are involved in viral replication and pathogenesis, whereas the three structural proteins are assembled into the mature virus particle.The E protein is the major surface protein of the flavivirus virions. The E protein is also the primary immunogen and plays a central role in virus attachment and entry to cells via membrane fusion. The x-ray crystallographic structures of the E protein ectodomains of both the tick-borne central European encephalitis virus (5) and the mosquito-borne dengue-2 virus (6) have been solved. Both proteins contain three distinct structural domains (domains I, II, and III) that correspond to previously characterized antigenic domains (7). Domain III (D3) of the E protein was initially proposed as the likely receptorbinding domain of the flaviviruses because of its structural characteristics. These include an IgC-like fold and a four-amino-acid loop that contains an RGD integrin-binding motif in several of the mosquito-borne flaviviruses (5). More recent studies have shown that D3 is directly associated with binding of dengue-2 virus (DEN2V) (8), WNV, and the tick-borne Langat virus 2 to cells. Domain 3 of WNV (WN-rED3) has also been shown to contain epitopes recognized by virus-neutralizing monoclonal antibodies (9). X-ray crystallography (5, 6) and cryo-electron microscopy studies (6, 10, 11) of the mosquitoborne WNV, DEN2V, and yellow fever virus have found that the E protein is arranged in dimeric form on the virus surface. Located at the 5-and 3-fold axes of symmetry, D3 projects...

Dynorphin A (1-17) (dynorphin) acts preferentially and with high affinity at the kappa-opioid receptor, for which it is the natural, endogenous ligand. Interest in designing new ligands to interact at the kappa-opioid receptor is based in part on the desire to circumvent some of the problems associated with mu-opioid ligands such as morphine. The high-resolution structure of dynorphin in an environment which closely resembles its environment in vivo could be considered as an important lead for new drugs. The interactions that occur between dynorphin and a model membrane are potentially important, as peptide hormone activity is thought to be mediated by interactions with the cell membrane. Therefore, we have determined the high-resolution structures of dynorphin in a model membrane. Results from our laboratory have shown the existence of an alpha-helical region in dynorphin from residues Gly3 through Arg9 when bound to perdeuterated dodecylphosphocholine (DPC) micelles. In this report we show that dynorphin is bound to DPC micelles and describe a family of dynorphin structures that is alpha-helical from residues Gly3 through Pro10 and that contains a beta-turn from residues Trp14 through Gln17. A model of interaction with the micelle is also reported and is discussed in the context of hormone action in vivo. The structures were determined with 1D and 2D nuclear magnetic resonance spectroscopy, distance geometry in dihedral angle space, and restrained molecular dynamics simulations.

No abstract

DNA molecules with covalently sealed (hairpin) ends are probable intermediates in V(D)J recombination. According to current models hairpin ends are opened to produce short single-stranded extensions that are thought to be precursors of a particular type of extra nucleotides, termed P nucleotides, which are frequently present at recombination junctions. Nothing is known about the activites responsible for hairpin opening. We have used two single-strand-specIfIc nucleases to explore the effects of loop sequence on the hairpin opening reaction. Here we show that a variety of hairpin ends are opened by P1 nuclease and mung bean nuclease (MBN) to leave short, 1-2 nt single-stranded extensions. Analysis of 22 dIfferent hairpin sequences demonstrates that the terminal 4 nt of the hairpin loop strongly influence the sites of cleavage. Correlation of the nuclease digestion patterns with structura (NMR) data for some of the hairpin loops studied here provides new insights into the structural features recognized by these enzymes.

No abstract

Calbindin D 28k , a member of the troponin C superfamily of calcium-binding proteins, contains six putative EF hand domains but binds only four calcium-atoms: one at a binding site of very high affinity and three calcium-atoms at binding sites of lower affinity. The high-affinity site could be located within domain I while domains III, IV, and V bind calcium less tightly. The recombinant protein construct calb I-II (residues 1±93) comprising the first two EF hands affords a unique opportunity to study a pair of EF hands with one site binding calcium tightly and the second site empty. A series of heteronuclear 2D, 3D and 4D high-resolution NMR experiments were applied to calb I-II, and led to the complete assignment of the 1 H, 13 C and 15 N resonances. The secondary structure of the protein was deduced from the size of the 3 J HN-Ha coupling constants, the chemical shift indices of 1 H a , 13 C a , 13 C H and 13 C b nuclei and from an analysis of backbone NOEs observed in 3D and 4D NOESY spectra. Four major a-helices are identified: Ala13±Phe23, Gly33±Ala50, Leu54±Asp63, Val76±Leu90, while residues Ala2±Leu6 form a fifth, flexible helical segment. Two short b-strands (Tyr30±Glu32, Lys72±Gly74) are found preceding helices B and D and are arranged in an anti-parallel interaction. Based on these data a structural model of calb I-II was constructed that shows that the construct adopts a tertiary structure related to other well-described calcium-binding proteins of the EF-hand family. Surprisingly, the protein forms a homodimer in solution, as was shown by its NMR characterization, size-exclusion chromatography and analytical ultra-centrifugation studies.Keywords: calbindin D 28k ; calcium binding protein; EF hand; NMR spectroscopy; secondary structure.Calbindin D 28k is an intracellular calcium binding protein of molecular mass 28 kDa that may be involved in transcellular calcium transport and may modulate effects occurring in response to changes in intracellular calcium concentrations. It was originally purified from chicken intestine [1] and is also expressed in kidney cells. The presence of the protein in numerous additional avian and mammalian tissues suggests an important physiological function [2]. The finding that it is particularly abundant in specific brain regions has led to studies investigating its particular role in calcium homeostasis [3,4].Calbindin D 28k belongs to a family of calcium-binding proteins including many well-known proteins such as calbindin D 9k , troponin C, S100B, calmodulin and calretinin. They are structurally characterized by one or more pairs of a helix-loophelix motif called the EF hand [5] in which five hydrophilic residues and one water molecule bind one calcium atom in a pentagonal bipyramidal coordination via oxygen atoms. The calcium-binding loop is flanked by two a-helices which have a number of residues with hydrophobic side chains that interact with a second EF hand domain forming a hydrophobic core. An approximate twofold symmetry axis relates the pair of EF hand domains....

The compound c[Cys5,11]dynorphin A-(1-11)-NH2, 1, is a cyclic dynorphin A analog that shows similar selectivity and potency at the kappa-opioid receptor when compared to the native form of the peptide in central nervous system assays. Previous molecular mechanics calculations have shown that the ring portion of the isoform that is trans about the Arg9-Pro10 omega bond contains either a beta-turn from residues Arg6 to Arg9 or an alpha-helical conformation. Our results from solution state NMR indicate that the compound exhibits cis-trans isomerism about the Arg9-Pro10 omega bond in both aqueous solution and when bound to dodecylphosphocholine micelles. Restrained molecular dynamics calculations show that the cis isoform of the peptide contains a type III beta-turn from residues Arg7 to Pro10. Similar calculations on the trans isoform show it to contain a beta-turn from residues Cys5 and Arg8. In this report we describe the generation of three-dimensional models from NMR data for the ring portions of both the cis and trans isoforms of 1 bound to dodecylphosphocholine micelles. Comparison with other dynorphin A structural information indicates that both the cis and trans isoforms of the peptide may be active as kappa-opioid agonists.