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Abstract In mitosis, accurate chromosome segregation depends on kinetochores that connect centromeric chromatin to spindle microtubules. The centromeres of budding yeast, which are relatively simple, are connected to individual microtubules via a kinetochore constitutive centromere associated network (CCAN). However, the complex centromeres of human chromosomes comprise millions of DNA base pairs and attach to multiple microtubules. Here, by use of cryo-electron microscopy and functional analyses, we reveal the molecular basis of how human CCAN interacts with duplex DNA and facilitates accurate chromosome segregation. The overall structure relates to the cooperative interactions and interdependency of the constituent sub-complexes of the CCAN. The duplex DNA is topologically entrapped by human CCAN. Further, CENP-N does not bind to the RG-loop of CENP-A but to DNA in the CCAN complex. The DNA binding activity is essential for CENP-LN localization to centromere and chromosome segregation during mitosis. Thus, these analyses provide new insights into mechanisms of action underlying kinetochore assembly and function in mitosis.

Uncovering conserved 3D protein–ligand binding patterns on the basis of functional groups (FGs) shared by a variety of small molecules can greatly expand our knowledge of protein–ligand interactions. Despite that conserved binding patterns for a few commonly used FGs have been reported in the literature, large-scale identification and evaluation of FG-based 3D binding motifs are still lacking. Here, we propose a computational method, Automatic FG-based Three-dimensional Motif Extractor (AFTME), for automatic mapping of 3D motifs to different FGs of a specific ligand. Applying our method to 233 naturally-occurring ligands, we define 481 FG-binding motifs that are highly conserved across different ligand-binding pockets. Systematic analysis further reveals four main classes of binding motifs corresponding to distinct sets of FGs. Combinations of FG-binding motifs facilitate the binding of proteins to a wide spectrum of ligands with various binding affinities. Finally, we show that our FG–motif map can be used to nominate FGs that potentially bind to specific drug targets, thus providing useful insights and guidance for rational design of small-molecule drugs.

Melatonin receptors (MT1 and MT2) are the targets for melatonin, the major neurohormone involved in circadian rhythm and sleep regulation. Here the authors describe the structures of 2-iodomelatonin and ramelteon bound MT1–Gi and MT2-Gi, revealing that MT1 and MT2 possess distinctive features within the ligand-binding pocket.

Abstract The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation. Trial registration: Clinicaltrials.gov NCT01588132.

Intracellular fatty acid-binding proteins are evolutionarily highly conserved proteins. The major functions and responsibilities of this family are the regulation of FA uptake and intracellular transport. The structure of the H-FABP ortholog from mouse (Mus musculus) had not been revealed at the time this study was completed. Thus, further exploration of the structural properties of mouse H-FABP is expected to extend our knowledge of the model animal’s molecular mechanism of H-FABP function. Here, we report the high-resolution crystal structure and the NMR characterization of mouse H-FABP. Our work discloses the unique structural features of mouse H-FABP, offering a structural basis for the further development of small-molecule inhibitors for H-FABP.

Abstract Background As hybrid RNAs, transcription-induced chimeras (TICs) may have tumor-promoting properties, and some specific chimeras have become important diagnostic markers and therapeutic targets for cancer. Methods We examined 23 paired laryngeal cancer (LC) tissues and adjacent normal mucous membrane tissue samples (ANMMTs). Three of these pairs were used for comparative transcriptomic analysis using high-throughput sequencing. Furthermore, we used real-time polymerase chain reaction (RT-PCR) for further validation in 20 samples. The Kaplan-Meier method and Cox regression model were used for the survival analysis. Results We identified 87 tumor-related TICs and found that COL7A1-UCN2 had the highest frequency in LC tissues (13/23; 56.5%), whereas none of the ANMMTs were positive (0/23; p

Identifying conserved binding motifs is an efficient way to study protein–ligand recognition. Most 3D binding motifs only contain information from the protein side, and so motifs that combine information from both protein and ligand sides are desired. Here, we propose an algorithm called LibME (Ligand‐binding Motif Extractor), which automatically extracts 3D binding motifs composed of the target ligand and surrounding conserved residues. We show that the motifs extracted by LibME for ATP and its analogs are highly similar to well‐known motifs reported by previous studies. The superiority of our method to handle flexible ligands was also demonstrated using isocitric acid as an example. Finally, we show that these motifs, together with their visual exhibition, permit better investigating and understanding of protein–ligand recognition process.

Human PRS1, which is indispensable for the biosynthesis of nucleotides, deoxynucleotides and their derivatives, is associated directly with multiple human diseases because of single base mutation. However, a molecular understanding of the effect of these mutations is hampered by the lack of understanding of its catalytic mechanism. Here, we reconstruct the 3D EM structure of the PRS1 apo state. Together with the native stain EM structures of AMPNPP, AMPNPP and R5P, ADP and the apo states with distinct conformations, we suggest the hexamer is the enzymatically active form. Based on crystal structures, sequence analysis, mutagenesis, enzyme kinetics assays, and MD simulations, we reveal the conserved substrates binding motifs and make further analysis of all pathogenic mutants.

Arginine methylation plays vital roles in the cellular functions of the protozoan Trypanosoma brucei. The T. brucei arginine methyltransferase 6 (TbPRMT6) is a type I arginine methyltransferase homologous to human PRMT6. In this study, we report the crystal structures of apo-TbPRMT6 and its complex with the reaction product S-adenosyl-homocysteine (SAH). The structure of apo-TbPRMT6 displays several features that are different from those of type I PRMTs that were structurally characterized previously, including four stretches of insertion, the absence of strand β15, and a distinct dimerization arm. The comparison of the apo-TbPRMT6 and SAH-TbPRMT6 structures revealed the fine rearrangements in the active site upon SAH binding. The isothermal titration calorimetry results demonstrated that SAH binding greatly increases the affinity of TbPRMT6 to a substrate peptide derived from bovine histone H4. The western blotting and mass spectrometry results revealed that TbPRMT6 methylates bovine histone H4 tail at arginine 3 but cannot methylate several T. brucei histone tails. In summary, our results highlight the structural differences between TbPRMT6 and other type I PRMTs and reveal that the active site rearrangement upon SAH binding is important for the substrate binding of TbPRMT6.

A study analyzes the case of a large Chinese family with X-linked nonsyndromic hearing loss (NSHL) and identifies missense mutations in the PRPS1 gene of the family members. It demonstrates that the mutations cause loss of function in the enzyme phosphoribosyl pyrophosphate (PRPP) synthetase 1, thereby resulting in the X-linked sensorineural deafness, which involves the DFN2 locus.

Crystal structure and mutagenesis analysis of the yGGA2_GAE in the domain of GAE proteins (Ent3 and Ent5) in Saccharomyces cerevisiae are performed to understand the domain binding mechanism of Ent3/5. The results suggested that Ent3 and Ent5 play a distinct role in trafficking by fine tuning the GGA adaptor dependence by adjusting their non-acidic-phenylalanine residues.

The crystal structure of the human nascent polypeptide-associated complex (NAC) domain was characterized to demonstrate the kinetics of human NAC dimerization. The results identified a region in the NAC domain of the human NAC [alpha]-subunit as a new nucleic acid-binding region which is blocked from binding nucleic acids in the heterodimeric complex by a helix region in the [beta]-subunit.

Natrin purified from Naja atra venom, a member of the cysteine-rich secretory proteins (CRISP) family, can induce a contractile response in the endothelium-denuded thoracic aorta of mouse which are contracted by a high-K(super +) solution. The experiments show that it could block the high-conductance calcium-activated potassium (BK(sub Ca)) channel that suggest that only a single natrin molecule is required to bind an ion channel to block BK(sub a) current.

UbiG and Coq3 (orthologue in eukaryotes) are SAM-MTases (Sadenosylmethionine- dependent methyltransferases) that catalyse both O-methylation steps in CoQ biosynthesis from prokaryotes to eukaryotes. However, the detailed molecular mechanism by which they function remains elusive. In the present paper, we report that UbiG/Coq3 defines a novel class of membrane-binding proteins. Escherichia coli UbiG binds specifically to liposomes containing PG (phosphatidylglycerol) or CL (cardiolipin, or diphosphatidylglycerol), two major lipid components of the E. coli plasma membrane, whereas human and yeast Coq3 display a strong preference for liposomes enriched with CL, a signature lipid of the mitochondrial membrane. The crystal structure of UbiG from E. coli was determined at 2.1 Å (1 Å = 0.1 nm) resolution. The structure exhibits a typical Class I SAM-MTase fold with several variations, including a unique insertion between strand β5 and helix α 10. This insertion is highly conserved and is required for membrane binding. Mutation of the key residues renders UbiG unable to efficiently bind liposome in vitro and the mutant fails to rescue the phenotype of ΓubiG strain in vivo. Taken together, our results shed light on a novel biochemical function of the UbiG/Coq3 protein. [ABSTRACT FROM AUTHOR]

Motivation: A large number of proteins contain metal ions that are essential for their stability and biological activity. Identifying and characterizing metal-binding sites through computational methods is necessary when experimental clues are lacking. Almost all published computational methods are designed to distinguish metal-binding sites from non-metal-binding sites. However, discrimination between different types of metal-binding sites is also needed to make more accurate predictions. Results: In this work, we proposed a novel algorithm called mFASD, which could discriminate different types of metal-binding sites effectively based on 3D structure data and is useful for accurate metal-binding site prediction. mFASD captures the characteristics of a metal-binding site by investigating the local chemical environment of a set of functional atoms that are considered to be in contact with the bound metal. Then a distance measure defined on functional atom sets enables the comparison between different metal-binding sites. The algorithm could discriminate most types of metal-binding sites from each other with high sensitivity and accuracy. We showed that cascading our method with existing ones could achieve a substantial improvement of the accuracy for metal-binding site prediction. [ABSTRACT FROM AUTHOR]

Yeast Hifl [Hatl (histone acetyltransferase 1)-interacting factor], a homologue of human NASP (nuclear autoantigenic sperm protein), is a histone chaperone that is involved in various protein complexes which modify histones during telomeric silencing and chromatin reassembly. For elucidating the structural basis of Hif1, in the present paper we demonstrate the crystal structure of Hifl consisting of a superhelixed TPR (tetratricopeptide repeat) domain and an extended acid loop covering the rear of TPR domain, which represent typical characteristics of SHNi-TPR [Sim3 (start independent of mitosis 3)-Hif1-NASP interrupted TPR] proteins. Our binding assay indicates that Hif1 could bind to the histone octamer via histones H3 and H4. The acid loop is shown to be crucial for the binding of histones and may also change the conformation of the TPR groove. By binding to the core histone complex Hif1 may recruit functional protein complexes to modify histones during chromatin reassembly. [ABSTRACT FROM AUTHOR]

Human CENP-N and CENP-L have been reported to selectively recognize the CENP-A nucleosome and to contribute to recruiting other constitutive centromere-associated network (CCAN) complexes involved in assembly of the inner kinetochore. As their homologues, Chl4 and Iml3 from budding yeast function in a similar way in de novo assembly of the kinetochore. A lack of biochemical and structural information precludes further understanding of their exact role at the molecular level. Here, the crystal structure of lml3 is presented and the structure shows that Iml3 adopts an elongated conformation with a series of intramolecular interactions. Pull-down assays revealed that the C-terminal domain of Chl4, which forms a dimer in solution, is responsible for Iml3 binding. Acting as a heterodimer, the Chl4-Iml3 complex exhibits a low-affinity nonspecific DNA-binding activity which may play an important role in the kinetochore-assembly process. [ABSTRACT FROM AUTHOR]

ArfGAP With Coiled-Coil, Ankyrin Repeat And PH Domains 4 (ACAP4) is an ADP-ribosylation factor 6 (ARF6) GTPase-activating protein essential for EGF-elicited cell migration. However, how ACAP4 regulates membrane dynamics and curvature in response to EGF stimulation is unknown. Here, we show that phosphorylation of the N-terminal region of ACAP4. named the Bin, Amphiphysin, and RSV161/167 (BAR) domain, at Tyr34 is necessary for EGF-elicited membrane remodeling. Domain structure analysis demonstrates that the BAR domain regulates membrane curvature. EGF stimulation of cells causes phosphorylation of ACAP4 at Tyr34. which subsequently promotes ACAP4 homodimer curvature. The phospho-mimicking mutant of ACAP4 demonstrates lipid-binding activity and tubulation in vitro, and ARF6 enrichment at the membrane is associated with ruffles of EGF-stimulated cells. Expression of the phospho-mimicking ACAP4 mutant promotes ARF6-dependent cell migration. Thus, the results present a previously undefined mechanism by which EGF-elicited phosphorylation of the BAR domain controls ACAP4 molecular plasticity and plasma membrane dynamics during cell migration. [ABSTRACT FROM AUTHOR]

The cleavage factor Im (CF Im), consists of a 25 kDa subunit (CF Im25) and one of three larger subunits (CF Im59, CF Im68, CF Im72), and is an essential protein complex for pre-mRNA 3′-end cleavage and polyadenylation. It recognizes the upstream sequence of the poly(A) site in a sequence-dependent manner. Here we report the crystal structure of human CF Im, comprising CF Im25 and the RNA recognition motif domain of CF Im68 (CF Im68RRM), and the crystal structure of the CF Im-RNA complex. These structures show that two CF Im68RRM molecules bind to the CF Im25 dimer via a novel RRM-protein interaction mode forming a heterotetramer. The RNA-bound structure shows that two UGUAA RNA sequences, with anti-parallel orientation, bind to one CF Im25-CF Im68RRM heterotetramer, providing structural basis for the mechanism by which CF Im binds two UGUAA elements within one molecule of pre-mRNA simultaneously. Point mutation and kinetic analyses demonstrate that CF Im68RRM can bind the immediately flanking upstream region of the UGUAA element, and CF Im68RRM binding significantly increases the RNA-binding affinity of the complex, suggesting that CF Im68 makes an essential contribution to pre-mRNA binding. [ABSTRACT FROM AUTHOR]

The adipocyte is the principal cell type for fat storage. CPT1 (carnitine palmitoyltransferase-1) is the rate-limiting enzyme for fatty acid β-oxidation, but the physiological role of CPT1 in adipocytes remains unclear. In the present study, we focused on the specific role of CPT1A in the normal functioning of adipocytes. Three 3T3-L1 adipocyte cell lines stably expressing hCPT1A (human CPT1A) cDNA, mouse CPT1A shRNA (short-hairpin RNA) or GFP (green fluorescent protein) were generated and the biological functions of these cell lines were characterized. Alteration in CPT1 activity, either by ectopic overexpression or pharmacological inhibition using etomoxir, did not affect adipocyte differentiation. However, overexpression of hCPT1A significantly reduced the content of intracellular NEFAs (non-esterified fatty acids) compared with the control cells when adipocytes were challenged with fatty acids. The changes were accompanied by an increase in fatty acid uptake and a decrease in fatty acid release. Interestingly, CPT1A protected against fatty acid-induced insulin resistance and expression of pro-inflammatory adipokines such as TNF-α (tumour necrosis factor-α) and IL-6 (interleukin-6) in adipocytes. Further studies demonstrated that JNK (c-Jun N terminal kinase) activity was substantially suppressed upon CPT1A overexpression, whereas knockdown or pharmacological inhibition of CPT1 caused a significant enhancement of JNK activity. The specific inhibitor of JNK SP600125 largely abolished the changes caused by the shRNA- and etomoxir-mediated decrease in CPT1 activity. Moreover, C2C12 myocytes co-cultured with adipocytes pre-treated with fatty acids displayed altered insulin sensitivity. Taken together, our findings have identified a favourable role for CPT1A in adipocytes to attenuate fatty acid-evoked insulin resistance and inflammation via suppression of JNK. [ABSTRACT FROM AUTHOR]

The snake-venom thrombin-like enzymes (SVTLEs) are a class of serine proteinases that show fibrinogen-clotting and esterolytic activities. Most TLEs convert fibrinogen to fibrin by releasing either fibrinopeptide A or fibrinopeptide B and cannot activate factor XIII. The enzymes hydrolyze fibrinogen to produce non-cross-linked fibrins, which are susceptible to the lytic action of plasmin. Because of these physiological properties, TLEs have important medical applications in myocardial infarction, ischaemic stroke and thrombotic diseases. Here, a three-step chromatography procedure was used to purify saxthrombin (AAP20638) from Gloydius saxatilis venom to homogeneity. Its molecular weight is about 30 kDa as estimated by SDS–PAGE. A saxthrombin crystal was obtained using the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 1.43 Å. The crystal belongs to space group C2, with unit-cell parameters a = 97.23, b = 52.21, c = 50.10 Å, β = 96.72°, and the Matthews coefficient ( VM) was calculated to be 2.13 Å3 Da−1 with one molecule in the asymmetric unit. [ABSTRACT FROM AUTHOR]

The fifth HMG-box domain in human upstream binding factor (hUBF) contributes to the synthesis of rRNA by RNA polymerase I (Pol I). The 2.0 Å resolution crystal structure of this protein has been solved using the single-wavelength anomalous dispersion method (SAD). The crystal structure and the reported NMR structure have r.m.s. deviations of 2.18–3.03 Å for the Cα atoms. However, there are significant differences between the two structures, with displacements of up to 9.0 Å. Compared with other HMG-box structures, the r.m.s. deviations for Cα atoms between hUBF HMG box 5 and HMG domains from Drosophila melanogaster protein D and Rattus norvegicus HMG1 are 1.5 and 1.6 Å, respectively. This indicates that the differences between the crystal and NMR structures of hUBF HMG box 5 are larger than those with its homologous structures. The differences between the two structures potentially reflect two states with different structures. The specific interactions between the hUBF HMG box 5 and the first bromodomain of TBP-associated factor 1 (TAF1) were studied by ultrasensitive differential scanning calorimetry and chemical shift perturbation. Based on these experimental data, possible sites in hUBF HMG box 5 that may interact with the first bromodomain of TAF1 were proposed. [ABSTRACT FROM AUTHOR]

Stecrisp from Trimeresurus stejnegeri snake venom belongs to a family of cysteine-rich secretory proteins (CRISP) that have various functions related to sperm-egg fusion, innate host defense, and the blockage of ion channels. Here we present the crystal structure of stecrisp refined to 1.6-Å resolution. It shows that stecrisp contains three regions, namely a PR-1 (pathogenesis-related proteins of group1) domain, a hinge, and a cysteine-rich domain (CRD). A conformation of solvent-exposed and -conserved residues (His60, Glu75, Glu96, and His115) in the PR-1 domain similar to that of their counterparts in homologous structures suggests they may share some molecular mechanism. Three flexible loops of hypervariable sequence surrounding the possible substrate binding site in the PR-1 domain show an evident difference in homologous structures, implying that a great diversity of species- and substrate-specific interactions may be involved in recognition and catalysis. The hinge is fixed by two crossed disulfide bonds formed by four of ten characteristic cysteines in the carboxyl-terminal region and is important for stabilizing the N- terminal PR-1 domain. Spatially separated from the PR-1 domain, CRD possesses a similar fold with two K+ channel inhibitors (Bgk and Shk). Several candidates for the possible functional sites of ion channel blocking are located in a solvent-exposed loop in the CRD. The structure of stecrisp will provide a prototypic architecture for a structural and functional exploration of the diverse members of the CRISP family. [ABSTRACT FROM AUTHOR]

The crystallization and preliminary crystallographic analysis of agkicetin-C, a well known platelet glycoprotein Ib (GPIb) antagonist from the venom of Deinagkistrodon acutus found in Anhui Province, China is reported. Crystals of agkicetin-C suitable for structure determination were obtained from 1.8 M ammonium sulfate, 40 mM MES pH 6.5 with 2%(v/v) PEG 400. Interestingly, low buffer concentrations of MES seem to be necessary for crystal growth. The crystals of agkicetin-C belong to space group C2, with unit-cell parameters a = 177.5, b = 97.7, c = 106.8 Å, β = 118.5°, and diffract to 2.4 Å resolution. Solution of the phase problem by the molecular-replacement method shows that there are four agkicetin-C molecules in the asymmetric unit, with a VM value of 3.4 ų Da-1, which corresponds to a high solvent content of approximately 64%. Self-rotation function calculations show a single well defined non-crystallographic twofold axis with features that may represent additional elements of non-crystallographic symmetry. [ABSTRACT FROM AUTHOR]

Abstract SPE10 is an antifungal protein isolated from the seeds of Pachyrrhizus erosus. cDNA encoding a 47 amino acid peptide was cloned by RT-PCR and the gene sequence proved SPE10 to be a new member of plant defensin family. The synthetic cDNA with codons preferred in yeast was cloned into the pPIC9 plasmid directly in-frame with the secretion signal a-mating factor, and highly expressed in methylotrophic Pichia pastoris. Activity assays showed the recombinant SPE10 inhibited specifically the growth of several pathogenic fungi as native SPE10. Circular dichroism and fluorescence spectroscopy analysis indicated that the native and recombinant protein should have same folding, though there are eight cystein residues in the sequence. Several evidence suggested SPE10 should be the first dimeric plant defensin reported so far. [ABSTRACT FROM AUTHOR]

Cysteine-rich secretory proteins (CRISPs) are widely distributed in mammals and snake venoms. They possess apparent homology but varying functions. The structure of CRISPs has remained elusive. Two novel members of the family, natrin and stecrisp, have been purified from Naja atra and Trimeresurus stejnegeri venoms, respectively. Their crystals diffract X-rays to resolution limits of 2.1 and 1.6 Å, repectively, and belong to the orthorhombic system with different space groups, unit-cell parameters and numbers of molecules per asymmetric unit. Their structures will contribute a structural basis for further functional studies of this family. [ABSTRACT FROM AUTHOR]

Atratoxin-b, a short-chain α-neurotoxin purified from Naja atra (mainland Chinese cobra) venom using a three-step chromatography procedure, has an apparent molecular mass of 6950 Da with an alkaline pI value (> 9.5) and consists of one single polypeptide chain as estimated by MALDI-TOF mass spectrometry and SDS-PAGE. The protein is toxic to mice, with an in vitro LD[SUB50] of about 0.18 mgkg[SUP-1]. Its N-terminal amino-acid sequence, LECHNQQSSQTPTIT, displays a very high homology to those of other α-neurotoxins. The overall three-dimensional structure of atratoxin-b is very similar to that of the homologous erabutoxin-a, as shown by the crystallographic molecular replacement and preliminary refinement results, with an R factor and R[SUBfree] of 27 and 29%, respectively. The microcrystal slowly grew to dimensions of approximate 0.1 × 0.1 × 0.15 mm over eight months using hanging-drop vapour-diffusion method. It gave a set of diffraction data to 1.56 Å resolution using X-rays of wavelength 1.1516 Å generated by the X-ray Diffraction and Scattering Station of beamline U7B at the National Synchrotron Radiation Laboratory (Hefei, China); this is the first example of the use of this beamline in protein crystallography. The crystals belong to the tetragonal space group P4[SUB1]2[SUB1]2, with unit-cell parameters a = 49.28 c = 44.80 Å, corresponding to one molecule per asymmetric unit and a volume-to-mass ratio of 1.96 Å[SUP3] Da[SUP-1]. [ABSTRACT FROM AUTHOR]

A new coagulation factor IX binding protein, AHP IX-bp, has been purified from Agkistrodon halys Pallas venom and estimated to be an AB heterodimer of about 25 kDa consisting of two chains (an A chain of 15.5 kDa and a B chain of 15 kDa) linked by one or more disulfide bonds. The N-terminal sequence of AHP IX-bp has been determined and aligned with C-type lectin-like proteins. The protein has a high sequence similarity to some snake-venom C-type lectin-like proteins. AHP IX-bp binds to coagulation factor IX but not to coagulation factor X. Moreover, AHP IX-bp shows binding to coagulation factor IX in both zinc ion-dependent and pH-dependent manners. The affinity between AHP IX-bp and coagulation factor IX is higher under neutral or weakly alkaline conditions than under weakly acidic conditions. Single crystals of AHP IX-bp grown at pH 6.5 and 7.5 diffract X-rays to 2.0 and 1.8 Å resolution, respectively. Both crystals are isomorphous and belong to the space group P1; only one AB heterodimer is present in the unit cell. [ABSTRACT FROM AUTHOR]

AaV-SP-I and AaV-SP-II, two glycosylated serine proteinases from Agkistrodon acutus venom with fibrinogenolysis and esterolysis activities, have been purified to homogeneity by three-step ionexchange chromatography. Estimated by SDS-PAGE, the molecular weights of AaV-SP-I and AaV-SP-II are about 32 and 31 kDa under reducing conditions and 26 and 25 kDa under non-reducing conditions, respectively. The first 24 N-terminal amino-acid residues are the same in both sequences and display a high homology with those of several snake-venom serine proteinases. However, the proteins possess obviously distinct carbohydrate contents. Using the conventional hanging-drop vapour-diffusion method, single crystals of both enzymes were grown that were suitable for X-ray diffraction analysis. The crystals of AaV-SP-I and AaV-SP-II belong to space groups P2[sub 1]2[sub 1]2[sub 1] and C2, respectively. In each case there is only one molecule in the asymmetric unit. [ABSTRACT FROM AUTHOR]

Atratoxin, a new α-neurotoxin purified to homogeneity by a series of liquid chromatographies from the venom of Naja naja atra (mainland Chinese cobra), is a small single-polypeptide alkaline protein with a pI of about 9.5 and molecular weight of 6952 Da estimated by mass spectrometry. Although the sequencing of the N-terminal 15 residues (LECHNQQTTQQPEGG) shows that this neurotoxic protein contains most of the residues, especially at the conserved positions, of the consensus sequence of short-chain α-neurotoxins, the natural mutations in the N-terminal Loop-1 presented by the sequence alignment may have structural or functional implications for the interactions between α-neurotoxins and related receptors. Single crystals of atratoxin have been grown from drops containing the necessary Cu2+ ions by the conventional hanging-drop vapour-diffusion method. The crystals diffract X-rays to 1.6 Å resolution and belong to space group C2221, with unit-cell parameters a = 47.36, b = 47.83, c = 91.31 Å, corresponding to a volume-to-mass ratio of 1.85 ų Da-1 and two molecules in each asymmetric unit. [ABSTRACT FROM AUTHOR]

A snake-venom C-type lectin-like protein, agkaggregin, has been isolated from Agkistrodon acutus venom. Agkaggregin has an apparent molecular mass of about 28 kDa and consists of two different types of subunits, an α-subunit (∼15 kDa) and a β-subunit (∼14 kDa). Agkaggregin has the ability to induce platelet aggregation at concentrations of the order of nanomoles. The agkaggre gin crystals grew for nearly a year by hanging-drop vapour diffusion and belong to the I222 space group, with unit-cell parameters a = 64.75, b = 74.21, c = 133.24 Å. One asymmetric unit contains one αβ heterodimer, corresponding to a volume-to-mass ratio of 2.795 ų Da-1. Agkaggregin may exist in two association forms: an αβ heterodimer and a dimer of αβ heterodimers that associates during the long process of crystallization. [ABSTRACT FROM AUTHOR]

Influenza A virus neuraminidase can be classified into groups 1 and 2 on the basis of its primary structure. The main structural feature of group 1 neuraminidase is an extra cavity in the active site, the 150-cavity. Here we present the crystal structure of neuraminidase from the 2009 pandemic H1N1 influenza strain. In contrast to other characterized N1 neuraminidases, which are all members of group 1, 2009 H1N1 neuraminidase does not have a 150-cavity. [ABSTRACT FROM AUTHOR]