Crystallization and preliminary X-ray diffraction analysis of importin-alpha acomplexed with NLS peptidomimetics

Importin-alpha is the nuclear import receptor that recognizes cargo proteins with nuclear localization sequences (NLSs). Tile study of NLS peptidomimetics can provide a better understanding of the requirements for the molecular recognition of cargo proteins by importin-alpha, and potentially engender a large number of applications in medicine. Importin-a was crystallized with a set of six NLS peptidomimetics, and X-ray diffraction data were collected in the range 2.1-2.5 angstrom resolution. Preliminary electron density calculations show that the ligands are present in the crystals. (c) 2005 Elsevier B.V All rights reserved.

Structural basis for the specificity of bipartite nuclear localization sequence binding by importin-α

Importin-alpha is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-alpha, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-alpha and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX10-12KRRK) that can be used to identify novel nuclear proteins.

Role of flanking sequences and phosphorylation in the recognition of the simian-virus-40 large T-antigen nuclear localization sequences by importin-a

The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser(112) in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-alpha (Impalpha), we co-crystallized non-autoinhibited Impalpha with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Impalpha. We confirm the important role of flanking sequences using binding assays. Unexpectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Impalpha. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impalpha, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impalpha.

Rugulotrosins A and B: Two new antibacterial metabolites from an Australian isolate of a penicillium sp.

Two new antibacterial agents, rugulotrosin A (1) and B (2), were obtained from cultures of a Penicillium sp. isolated from soil samples acquired near Sussex Inlet, New South Wales, Australia. Rugulotrosin A (1) is a chiral symmetric dimer, and its relative stereostructure was determined by spectroscopic and X-ray crystallographic analysis. Rugulotrosin B (2) is a chiral asymmetric dimer isomeric with 1. Its structure was determined by spectroscopic analysis with comparison to the co-metabolite 1 and previously reported fungal metabolites. Both rugulotrosins A and B displayed significant antibacterial activity against Bacillus subtilis, while rugulotrosin A was also strongly active against Enterococcus faecalis and B. cereus.

Post-crystallization treatments for improving diffraction quality of protein crystals

X-ray crystallography is the most powerful method for determining the three-dimensional structure of biological macromolecules. One of the major obstacles in the process is the production of high-quality crystals for structure determination. All too often, crystals are produced that are of poor quality and are unsuitable for diffraction studies. This review provides a compilation of post-crystallization methods that can convert poorly diffracting crystals into data-quality crystals. Protocols for annealing, dehydration, soaking and cross-linking are outlined and examples of some spectacular changes in crystal quality are provided. The protocols are easily incorporated into the structure-determination pipeline and a practical guide is provided that shows how and when to use the different post-crystallization treatments for improving crystal quality.

Synthesis, characterization and X-ray crystal structures of dinuclear nickel(II) complexes of a novel potentially hexadentate but functionally tetradentate binucleating ligand

A binucleating potentially hexadentate chelating agent containing oxygen, nitrogen and sulfur as potential donor atoms (H2ONNO) has been synthesized by condensing alpha,alpha-xylenebis(N-methyldithiocarbazate) with 2,4-pentanedione. An X-ray crystallographic structure determination shows that the Schiff base remains in its ketoimine tautomeric form with the protons attached to the imine nitrogen atoms. The reaction of the Schiff base with nickel(II) acetate in a 1:1 stoichiometry leads to the formation of a dinuclear nickel(II) complex [Ni(ONNO)](2) (ONNO2- = dianionic form of the Schiff base) containing N,O-chelated tetradentate ligands, the sulfur donors remaining uncoordinated. A single crystal X-ray structure determination of this dimer reveals that each ligand binds two low spin nickel(II) ions, bridged by a xylyl group. The nickel(II) atoms adopt a distorted square-planar geometry in a trans-N2O2 donor environment. Reaction of the Schiff base with nickel(II) acetate in the presence of excess pyridine leads to the formation of a similar dinuclear complex, [Ni(ONNO)(py)](2), but in this case comprises five coordinate high-spin Ni(II) ions with pyridine ligands occupying the axial coordination sites as revealed by X-ray crystallographic analysis. (c) 2005 Published by Elsevier B.V.

Functional recycling of C2 domains throughout evolution: A comparative study of synaptotagmin, protein kinase C and phospholipase C by sequence, structural and modelling approaches

The C2 domain is one of the most frequent and widely distributed calcium-binding motifs. Its structure comprises an eight-stranded beta-sandwich with two structural types as if the result of a circular permutation. Combining sequence, structural and modelling information, we have explored, at different levels of granularity, the functional characteristics of several families of C2 domains. At the coarsest level,the similarity correlates with key structural determinants of the C2 domain fold and, at the finest level, with the domain architecture of the proteins containing them, highlighting the functional diversity between the various subfamilies. The functional diversity appears as different conserved surface patches throughout this common fold. In some cases, these patches are related to substrate-binding sites whereas in others they correspond to interfaces of presumably permanent interaction between other domains within the same polypeptide chain. For those related to substrate-binding sites, the predictions overlap with biochemical data in addition to providing some novel observations. For those acting as protein-protein interfaces' our modelling analysis suggests that slight variations between families are a result of not only complementary adaptations in the interfaces involved but also different domain architecture. In the light of the sequence and structural genomic projects, the work presented here shows that modelling approaches along with careful sub-typing of protein families will be a powerful combination for a broader coverage in proteomics. (C) 2003 Elsevier Ltd. All rights reserved.

Identification and analysis of venom gland-specific genes from the coastal taipan (Oxyuranus scutellatus) and related species

Australian terrestrial elapid snakes contain amongst the most potently toxic venoms known. However, despite the well-documented clinical effects of snake bite, little research has focussed on individual venom components at the molecular level. To further characterise the components of Australian elapid venoms, a complementary (cDNA) microarray was produced from the venom gland of the coastal taipan (Oxyuranus scutellatus) and subsequently screened for venom gland-specific transcripts. A number of putative toxin genes were identified, including neurotoxins, phospholipases, a pseudechetoxin-like gene, a venom natriuretic peptide and a nerve growth factor together with other genes involved in cellular maintenance. Venom gland-specific components also included a calglandulin-like protein implicated in the secretion of toxins from the gland into the venom. These toxin transcripts were subsequently identified in seven other related snake species, producing a detailed comparative analysis at the cDNA and protein levels. This study represents the most detailed description to date of the cloning and characterisation of different genes associated with envenomation from Australian snakes.

Inhibitors of cyclo-oxygenase-2 and secretory phospholipase A2 preserve bone architecture following ovariectomy in adult rats

Epidemiological evidence and in vitro data suggest that COX-2 is a key regulator of accelerated remodeling. Accelerated states of osteoblast and osteoclast activity are regulated by prostaglandins in vitro, but experimental evidence for specific roles of cyclooxygenase-2 (COX-2) and secretory phospholipase A(2) (sPLA(2)) in activated states of remodeling in vivo is lacking. The aim of this study was to determine the effect of specific inhibitors of sPLA(2)-IIa and COX-2 on bone remodeling activated by estrogen deficiency in adult female rats. One hundred and twenty-four adult female Wistar rats were ovariectomized (OVX) or sham-operated. Rats commenced treatment 14 days after surgery with either vehicle, a COX-2 inhibitor (DFU at 0.02 mg/kg/day and 2.0 mg/kg/day) or a sPLA(2)-group-IIa inhibitor (KH064 at 0.4 mg/kg/day and 4.0 mg/kg/day). Treatment continued daily until rats were sacrificed at 70 days or 98 days post-OVX. The right tibiae were harvested, fixed and embedded in methylmethacrylate for structural histomorphometric bone analysis at the proximal tibial metaphysis. The specific COX-2 or sPLA(2) inhibitors prevented ovariectomy-induced (OVX-induced) decreases in trabecular connectivity (P < 0.05); suppressed the acceleration of bone resorption; and maintained bone turnover at SHAM levels following OVX in the rat. The sPLA2 inhibitor significantly suppressed increases in osteoclast surface induced by OVX (P < 0.05), while the effect of COX-2 inhibition was less marked. These findings demonstrate that inhibitors of COX-2 and sPLA(2)-IIa can effectively suppress OVX-induced bone loss in the adult rat by conserving trabecular bone mass and architecture through reduced bone remodeling and decreased resorptive activity. Moreover, we report an important role of sPLA(2)-IIa in osteoclastogenesis that may be independent of the COX-2 metabolic pathway in the OVX rat in vivo. (c) 2006 Elsevier Inc. All rights reserved.