The crystal structure of murine p97/VCP at 3.6A

p97/VCP is a member of the AAA ATPase family and has roles in both membrane fusion and ubiquitin dependent protein degradation. Here, we present a 3.6A crystal structure of murine p97 in which D2 domain has been modelled as poly-alanine and the remaining approximately 100 residues are absent. The resulting structure illustrates a head-to-tail packing arrangement of the two p97 AAA domains in a natural hexameric state with D1 ADP bound and D2 nucleotide free. The head-to-tail packing arrangement observed in this structure is in contrast to our previously predicted tail-to-tail packing model. The linker between the D1 and D2 domains is partially disordered, suggesting a flexible nature. Normal mode analysis of the crystal structure suggests anti-correlated motions and distinct conformational states of the two AAA domains.     

Structure and properties of avian small heat shock protein with molecular weight 25 kDa

The primary structure of chicken small heat shock protein (sHsp) with apparent molecular weight 25 kDa was refined and it was shown that this protein has conservative primary structure 74RALSRQLSSG(83) at Ser77 and Ser81, which are potential sites of phosphorylation. Recombinant wild-type chicken Hsp25, its three mutants, 1D (S15D), 2D (S77D+S81D) and 3D (S15D+S77D+S81D), as well as delR mutant with the primary structure 74RALS-ELSSG(82) at potential sites of phosphorylation were expressed and purified. It has been shown that the avian tissues contain three forms of Hsp25 having pI values similar to that of the wild-type protein, 1D and 2D mutants that presumably correspond to nonphosphorylated, mono- and di-phosphorylated forms of Hsp25. Recombinant wild-type protein, its 1D mutant and Hsp25, isolated from chicken gizzard, form stable high molecular weight oligomeric complexes. The delR, 2D and 3D mutants tend to dissociate and exist in the form of a mixture of high and low molecular weight oligomers. Point mutations mimicking phoshorylation decrease chaperone activity of Hsp25 measured by reduction of dithiothreitol induced aggregation of alpha-lactalbumin, but increase the chaperone activity of Hsp25 measured by heat induced aggregation of alcohol dehydrogenase. It is concluded that avian Hsp25 has a more stable quaternary structure than its mammalian counterparts and mutations mimicking phosphorylation differently affect chaperone activity of avian Hsp25, depending on the nature of target protein and the way of denaturing.     

RNA 3D Modules in Genome-Wide Predictions of RNA 2D Structure

Recent experimental and computational progress has revealed a large potential for RNA structure in the genome. This has been driven by computational strategies that exploit multiple genomes of related organisms to identify common sequences and secondary structures. However, these computational approaches have two main challenges: they are computationally expensive and they have a relatively high false discovery rate (FDR). Simultaneously, RNA 3D structure analysis has revealed modules composed of non-canonical base pairs which occur in non-homologous positions, apparently by independent evolution. These modules can, for example, occur inside structural elements which in RNA 2D predictions appear as internal loops. Hence one question is if the use of such RNA 3D information can improve the prediction accuracy of RNA secondary structure at a genome-wide level. Here, we use RNAz in combination with 3D module prediction tools and apply them on a 13-way vertebrate sequence-based alignment. We find that RNA 3D modules predicted by metaRNAmodules and JAR3D are significantly enriched in the screened windows compared to their shuffled counterparts. The initially estimated FDR of 47.0% is lowered to below 25% when certain 3D module predictions are present in the window of the 2D prediction. We discuss the implications and prospects for further development of computational strategies for detection of RNA 2D structure in genomic sequence.     

Secondary structure of human interleukin 2 from 3D heteronuclear NMR experiments.

Recombinant 15N-labeled human interleukin 2 (IL-2) has been studied by 2D and 3D NMR using uniformly 15N-labeled protein. Assignment of the backbone resonances has enabled the secondary structure of the protein to be defined. The secondary structure was found to consist of four alpha-helical regions and a short section of antiparallel beta-sheet. This structure is more similar to recent published structures of interleukin 4 and granulocyte-macrophage colony-stimulating factor than to a structure of IL-2 previously obtained from low-resolution X-ray diffraction data.     

Structure prediction and fold recognition for the ferrochelatase family of proteins

An α/β barrel is predicted for the three-dimensional (3D) structure of Bacillus subtilis ferrochelatase. To arrive at this structure, the THREADER program was used to find possible homologous 3D structures and to predict the secondary structure for the ferrochelatase sequence. The secondary structure was fit by hand to the selected homologous 3D structure then the MODELLER program was used to predict the fold of ferrochelatase. Molecular biological information about the conserved residues of ferrochelatase was used as the criteria to help select the homologous 3D structure used to predict the fold of ferrochelatase. Based on the predicted structure possible, ligands binding to the iron and protoporphyrin IX are discussed. The structure has been deposited in the Brookhaven database as ID 1FJI. © 1997 Wiley-Liss Inc.     

Crystal structure of fragment D from lamprey fibrinogen complexed with the peptide Gly-His-Arg-Pro-amide

The crystal structure of fragment D from lamprey fibrinogen has been determined at 2.8 A resolution. The 89 kDa protein was cocrystallized with the peptide Gly-His-Arg-Pro-amide, which in many fibrinogens-but not lamprey-corresponds to the B knob exposed by thrombin. Because lamprey fragment D is more than 50% identical in sequence with human fragment D, the structure of which has been reported previously, it was possible to use the method of molecular replacement. The space group of the lamprey crystals is P1; there are four molecules in the unit cell. Although the fragments are packed head to head by the same D:D interface as is observed in other related preparations containing fragments D, the tails are uniquely joined by an unnatural association of the terminal sections of the residual coiled coils from adjacent molecules. Some features of the lamprey structure are clearer than have been observed in previous fragment D structures, including the beta-chain carbohydrate cluster, for one, and the important gamma-chain carboxyl-terminal segment, for another. The most significant differences between the lamprey and human structures occur in connecting loops at the entryways to the beta-chain and gamma-chain binding pockets.     

The hypervariable D3 domain of Salmonella flagellin is an autonomous folding unit

The hypervariable D3 domain of Salmonella flagellin, composed of the 190-285 segment, is the major determinant of flagellar antigenicity. D3 was cloned and overexpressed in E. coli. Although previous studies concluded that D3 is stabilized by interactions with the D2 domain, our calorimetric experiments have revealed that isolated D3 has a stable tertiary structure which is highly resistant against proteolytic digestion. Repeated heating experiments demonstrated that unfolding of D3 is reversible. Its small size and stable structure makes D3 a promising protein scaffold for the development of artificial binding proteins by directed evolution.     

Structural characterisation of a highly branched exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB2074

Lactobacillus delbrueckii subsp. bulgaricus NCFB2074 when grown in skimmed milk secretes a highly branched exopolysaccharide. The exopolysaccharide has a heptasaccharide repeat unit and is composed of glucose and galactose in the molar ratio 3:4. Using chemical techniques and 1D and 2D NMR spectroscopy the polysaccharide has been shown to possess the following repeat unit structure: [carbohydrate structure: see text].     

Crystal structure of Escherichia coli RNase D, an exoribonuclease involved in structured RNA processing

RNase D (RND) is one of seven exoribonucleases identified in Escherichia coli. RNase D has homologs in many eubacteria and eukaryotes, and has been shown to contribute to the 3' maturation of several stable RNAs. Here, we report the 1.6 A resolution crystal structure of E. coli RNase D. The conserved DEDD residues of RNase D fold into an arrangement very similar to the Klenow fragment exonuclease domain. Besides the catalytic domain, RNase D also contains two structurally similar alpha-helical domains with no discernible sequence homology between them. These closely resemble the HRDC domain previously seen in RecQ-family helicases and several other proteins acting on nucleic acids. More interestingly, the DEDD catalytic domain and the two helical domains come together to form a ring-shaped structure. The ring-shaped architecture of E. coli RNase D and the HRDC domains likely play a major role in determining the substrate specificity of this exoribonuclease.     

若干种枝角类卵鞍表面亚显微结构的比较研究

以扫描电镜对发头裸腹溞、蚤状溞、隆线溞指名亚种以及隆线溞东湖一亚种的卵鞍分别进行了比较研究,发现卵鞍表面的亚显微结构有明显的差异,但亚种之间差异较小。隆线溞指名亚种与隆线溞东湖一亚种二者的卵鞍在光学显微镜下看不出有什么差异,然而亚显微结构却互不相同。这就为东湖一新亚种的确定提供了一项可靠的依据,同时也进一步证实卵鞍的超微结构确可作为枝角类分类的表征。     

Rhoiptelenol and rhoiptelenone, two pentacyclic triterpenes from Sideritis macrostachya

The pentacyclic triterpenes rhoiptelenol and rhoiptelenone have been isolated from Sideritis macrostachya. Rhoiptelenone is a new natural compound, whose structure has been determined as D:B-friedo-urs-5-en-3-one. The 1H and 13C NMR spectra of rhoiptelenol, rhoiptelenol acetate and glutinol have been reassigned. The natural occurrence of the D:B-friedo-ursene and D:B-friedo-oleanene derivatives has been examined.     

Polarized Fourier transform infrared spectroscopy of bacteriorhodopsin. Transmembrane alpha helices are resistant to hydrogen/deuterium exchange.

The secondary structure of bacteriorhodopsin has been investigated by polarized Fourier transform infrared spectroscopy combined with hydrogen/deuterium exchange, isotope labeling and resolution enhancement methods. Oriented films of purple membrane were measured at low temperature after exposure to H2O or D2O. Resolution enhancement techniques and isotopic labeling of the Schiff base were used to assign peaks in the amide I region of the spectrum. alpha-helical structure, which exhibits strong infrared dichroism, undergoes little H/D exchange, even after 48 h of D2O exposure. In contrast, non-alpha-helical structure, which exhibits little dichroism, undergoes rapid H/D exchange. A band at 1,640 cm-1, which has previously been assigned to beta-sheet structure, is found to be due in part to the C = N stretching vibration of protonated Schiff base of the retinylidene chromophore. We conclude that the membrane spanning regions of bR consist predominantly of alpha-helical structure whereas most beta-type structure is located in surface regions directly accessible to water.     

Optimal Interfacial Engineering with Different Length of Alkylammonium Halide for Efficient and Stable Perovskite Solar Cells

Recently, two‐dimensional (2D) structure on three‐dimensional (3D) perovskites (graded 2D/3D) has been reported to be effective in significantly improving both efficiency and stability. However, the electrical properties of the 2D structure as a passivation layer on the 3D perovskite thin film and resistance to the penetration of moisture may vary depending on the length of the alkyl chain. In addition, the surface defects of the 2D itself on the 3D layer may also be affected by the correlation between the 2D structure and the hole conductive material. Therefore, systematic interfacial study with the alkyl chain length of long‐chained alkylammonium iodide forming a 2D structure is necessary. Herein, the 2D interfacial layers formed are compared with butylammonium iodide (BAI), octylammonium iodide (OAI), and dodecylammonium iodide (DAI) iodide on a 3D (FAPbI₃)_0.95(MAPbBr₃)_0.05 perovskite thin film in terms of the PCE and humidity stability. As the length of the alkyl chain increased from BA to OA to DA, the electron‐blocking ability and humidity resistance increase significantly, but the difference between OA and DA is not large. The PSC post‐treated with OAI has slightly higher PCE than those treated with BAI and DAI, achieving a certified stabilized efficiency of 22.9%.     

Crystal structure of human cytochrome P450 2D6

Cytochrome P450 2D6 is a heme-containing enzyme that is responsible for the metabolism of at least 20% of known drugs. Substrates of 2D6 typically contain a basic nitrogen and a planar aromatic ring. The crystal structure of human 2D6 has been solved and refined to 3.0A resolution. The structure shows the characteristic P450 fold as seen in other members of the family, with the lengths and orientations of the individual secondary structural elements being very similar to those seen in 2C9. There are, however, several important differences, the most notable involving the F helix, the F-G loop, the B'helix, beta sheet 4, and part of beta sheet 1, all of which are situated on the distal face of the protein. The 2D6 structure has a well defined active site cavity above the heme group, containing many important residues that have been implicated in substrate recognition and binding, including Asp-301, Glu-216, Phe-483, and Phe-120. The crystal structure helps to explain how Asp-301, Glu-216, and Phe-483 can act as substrate binding residues and suggests that the role of Phe-120 is to control the orientation of the aromatic ring found in most substrates with respect to the heme. The structure has been compared with published homology models and has been used to explain much of the reported site-directed mutagenesis data and help understand the metabolism of several compounds.     

Picornavirus RNA-dependent RNA polymerase

Replication of the picornavirus genome is catalysed by a viral encoded RNA-dependent RNA polymerase, termed 3D polymerase. Together with other viral and host proteins, this enzyme performs its functions in the cytoplasm of host cells. The crystal structure of 3D polymerase from a number of picornaviruses has been determined. This review discusses the structure and function of the poliovirus 3D polymerase. The high error rates of 3D polymerase result in high sequence diversity such that virus populations exist as quasispecies. This phenomenon is thought to facilitate survival of the virus population in complex environments.     

Paradigm development: comparative and predictive 3D modeling of HIV-1 Virion Infectivity Factor (Vif)

Obtaining structural information about Vif is of interest for several reasons that include the study of the interaction of Vif with APOBEC3G, a resistance factor. Vif is a potential drug target and its function is essential for the HIV-1 infectivity process. To study Vif mechanism of action, we need to decipher its structure. Pivotal in this approach is the painstaking prediction of its protein structure. The three-dimensional (3D) crystal structure for Vif has not been established. In order to understand its mechanism of action, information on the structure of Vif is very much needed. Therefore we undertook this study based on the hypothesis that information from structurally homologous proteins can be used to predict the 3D structure of Vif by computer modeling and threading. As a result the structure of HIV-1 Vif has been modeled and deposited in the theoretical models section and accepted with the PDB code 1VZF. Here, we present the results of the comparative modeling strategy we used to predict the 3D structure of Vif.     

Crystal structure and conformational analysis of ampullosporin A.

Ampullosporin A is a 15-mer peptaibol type polypeptide that induces pigment formation by the fungus Phoma destructiva, forms voltage-dependent ion channels in membranes and exhibits hypothermic effects in mice. The structure of ampullosporin A has been determined by x-ray crystallography. This is the first three-dimensional (3D) structure of the peptaibol subfamily SF6. From the N-terminus to residue 13 the molecule adopts an approximate right-handed alpha-helical geometry, whereas a less regular structure pattern with beta-turn characteristics is found in the C-terminus. Even though ampullosporin A does not contain a single proline or hydroxyproline it is significantly bent. It belongs to both the shortest and the most strongly bent peptaibol 3D structures. The straight structure part encompasses residues Ac-Trp(1)-Aib(10) and is thus less extended than the alpha-helical subunit. The 3D structure of ampullosporin A is discussed in relation to other experimentally determined peptaibol structures and in the context of its channel-forming properties. As a part of this comparison a novel bending analysis based on a 3D curvilinear axis describing the global structural characteristics has been proposed and applied to all 3D peptaibol structures. A sampling of 2500 conformations using different molecular dynamics protocols yields, for the complete ampullosporin A structure, an alpha-helix as the preferred conformation in vacuo with almost no bend. This indicates that solvent or crystal effects may be important for the experimentally observed peptide backbone bending characteristics of ampullosporin A.     

A structural basis for the unique binding features of the human vitamin D-binding protein.

The human serum vitamin D-binding protein (DBP) has many physiologically important functions, ranging from transporting vitamin D3 metabolites, binding and sequestering globular actin and binding fatty acids to functioning in the immune system. Here we report the 2.3 A crystal structure of DBP in complex with 25-hydroxyvitamin D3, a vitamin D3 metabolite, which reveals the vitamin D-binding site in the N-terminal part of domain I. To more explicitly explore this, we also studied the structure of DBP in complex with a vitamin D3 analog. Comparisons with the structure of human serum albumin, another family member, reveal a similar topology but also significant differences in overall, as well as local, folding. These observed structural differences explain the unique vitamin D3-binding property of DBP.