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ABSTRACT: AhV_aPA, the acidic PLA2 purified from Agkistrodon halys pallas venom, was previously reported to possess a strong enzymatic activity and can remarkably induce a further contractile response on the 60 mM K(+)-induced contraction with an EC50 in 369 nM on mouse thoracic aorta rings. In the present study, we found that the p-bromo-phenacyl-bromide (pBPB), which can completely inhibit the enzymatic activity of AhV_aPA, did not significantly reduce the contractile response on vessel rings induced by AhV_aPA, indicating that the vasoconstrictor effects of AhV_aPA are independent of the enzymatic activity. The inhibitor experiments showed that the contractile response induced by AhV_aPA is mainly attributed to the Ca(2+) releasing from Ca(2+) store, especially sarcoplasmic reticulum (SR). Detailed studies showed that the Ca(2+) release from SR is related to the activation of inositol trisphosphate receptors (IP3Rs) rather than ryanodine receptors (RyRs). Furthermore, the vasoconstrictor effect could be strongly reduced by pre-incubation with hepain, indicating that the basic amino acid residues on the surface of AhV_aPA may be involved in the interaction between AhV_aPA and the molecular receptors. These findings offer new insights into the functions of snake PLA2 and provide a novel pathogenesis of A. halys pallas venom.
Toxicon 05/2013; · 2.51 Impact Factor
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ABSTRACT: Phosphorylated derivatives of phosphatidylinositol (PtdIns), also called phosphoinositides (PIPs), are basic components of membrane-associated signalling systems. A family of PtdIns-transfer proteins (PITPs) called the Sec14 family have been predicted to form a set of functional modules that can sense different types of lipid metabolism and transmit the information to the PIP signalling system. In eukaryotic cells, the Sec14 family exhibits a wide diversity of activity, but the structural basis of this diversity remains unclear. In the present study, the dimeric structure of Sfh3 (Sec14 family homologue 3 in yeast) is reported for the first time and differs from the Sec14 proteins reported to date, all of which are monomeric. Some variations in the binding pocket of Sfh3 were observed and the dimer interface was identified and proposed to provide a link between dimer-monomer state changes and PtdIns binding. Together, these structural changes and the oligomeric state transformation of Sfh3 support ideas of diversity within the Sec14 family and provide some new clues to function.
Acta crystallographica. Section D, Biological crystallography 03/2013; 69(Pt 3):313-23. · 12.67 Impact Factor
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ABSTRACT: Human RNA-binding protein (HuR), a ubiquitously expressed member of the Hu protein family, plays an important role in mRNA degradation and has been implicated as a key post-transcriptional regulator. HuR contains three RNA-recognition motif (RRM) domains. The two N-terminal tandem RRM domains can selectively bind AU-rich elements (AREs), while the third RRM domain (RRM3) contributes to interactions with the poly-A tail of target mRNA and other ligands. Here, the X-ray structure of two methylated tandem RRM domains (RRM1/2) of HuR in their RNA-free form was solved at 2.9 Å resolution. The crystal structure of RRM1/2 complexed with target mRNA was also solved at 2.0 Å resolution; comparisons of the two structures show that HuR RRM1/2 undergoes conformational changes upon RNA binding. Fluorescence polarization assays (FPA) were used to study the protein-RNA interactions. Both the structure and the FPA analysis indicated that RRM1 is the primary ARE-binding domain in HuR and that the conformational changes induce subsequent contacts of the RNA substrate with the inter-domain linker and RRM2 which greatly improve the RNA-binding affinity of HuR.
Acta crystallographica. Section D, Biological crystallography 03/2013; 69(Pt 3):373-80. · 12.67 Impact Factor
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Wei Zhao,
Wangsheng Chu,
Feifei Yang,
Meijuan Yu,
Dongliang Chen,
Xiaoyun Guo,
D. W. Zhou,
N. Shi,
August o Marcelli, Liwen Niu,
Maikun Teng,
Weimin Gong,
Maurizio Benfatto,
and Ziyu Wu
02/2013;
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ABSTRACT: Ap(4)A hydrolase (asymmetrical diadenosine tetraphosphate hydrolase, EC 3.6.1.17), an enzyme involved in a number of biological processes, is characterized as cleaving the polyphosphate chain at the fourth phosphate from the bound adenosine moiety. This paper presents the crystal structure of wild-type and E58A mutant human Ap(4)A hydrolase. Similar to the canonical Nudix fold, human Ap(4)A hydrolase shows the common αβα-sandwich architecture. Interestingly, two sulfate ions and one diphosphate coordinated with some conserved residues were observed in the active cleft, which affords a better understanding of a possible mode of substrate binding.
Biochemical and Biophysical Research Communications 02/2013; · 2.48 Impact Factor
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ABSTRACT: Phospholipases A(2) (PLA(2)s) are the major component of snake venoms and exert a variety of relevant toxic actions such as neurotoxicity and myotoxicity, amongst others. An acidic PLA(2), here named AhV_aPA, was purified from Agkistrodon halys pallas venom by means of a three-step chromatographic procedure. AhV_aPA migrated as a single band on SDS-PAGE gels, with a molecular weight of about 14 kDa. Like other acidic aPLA(2)s, AhV_aPA has high enzymatic activity. Tension measurements of mouse thoracic aortic rings remarkably indicated that AhV_aPA could induce a further contractile response on the 60 mM K(+)-induced contraction, with an EC(50) of 369 nmol l(-1). Rod-shaped crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 2.30 Å. The crystals belonged to space group P222, with unit-cell parameters a = 44.27, b = 68.39, c = 81.54 Å.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 11/2012; 68(Pt 11):1329-1332. · 0.51 Impact Factor
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ABSTRACT: A snake venom thrombin-like enzyme (SVTLE) from Agkistrodon halys pallas venom was isolated by means of a two-step chromatographic procedure. The purified enzyme, named AhV_TL-I, showed fibrinogenolytic activity against both the Aα and Bβ chains of bovine fibrinogen. Unlike the other SVTLEs, AhV_TL-I has poor esterolytic activity upon BAEE substrate. The N-terminal sequence of AhV_TL-I was determined to be IIGGDEXNINEHRFLVALYT, and the molecular mass was confirmed to 29389.533 Da by MALDI-TOF mass spectrometry. Its complete cDNA and derived amino acid sequence were obtained by RT-PCR. The crystal structure of AhV_TL-I was determined at a resolution of 1.75 Å. A disaccharide was clearly mapped in the structure, which involved in regulating the esterolytic activity of AhV_TL-I. The presence of the N-glycan deformed the 99-loop, and the resulting steric hindrances hindered the substrates to access the active site. Furthermore, with the carbohydrate moiety, AhV_TL-I could induce mouse thoracic aortic ring contraction with the EC(50) of 147 nmol/L. Besides, the vasoconstrictor effects of AhV_TL-I were also independent of the enzymatic activity. The results of [Ca(2+)](i) measurement showed that the vasoconstrictor effects of AhV_TL-I were attributed to Ca(2+) releasing from Ca(2+) store. Further studies showed that it was related to the activation of ryanodine receptors (RyRs). These offer new insights into the snake SVTLEs functions and provide a novel pathogenesis of A. halys pallas venom.
Archive für Toxikologie 10/2012; · 4.67 Impact Factor
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ABSTRACT: The yeast Paf1 complex (Paf1C), which is composed of the proteins Paf1, Cdc73, Ctr9, Leo1 and Rtf1, accompanies RNA polymerase II from the promoter to the 3'-end formation site of mRNA- and snoRNA-encoding genes. As one of the first identified subunits of Paf1C, yeast Cdc73 (yCdc73) takes part in many transcription-related processes, including binding to RNA polymerase II, recruitment and activation of histone-modification factors and communication with other transcriptional activators. The human homologue of yCdc73, parafibromin, has been identified as a tumour suppressor linked to breast, renal and gastric cancers. However, the functional mechanism of yCdc73 has until recently been unclear. Here, a 2.2 Å resolution crystal structure of the highly conserved C-terminal region of yCdc73 is reported. It revealed that yCdc73 appears to have a GTPase-like fold. However, no GTPase activity was observed. The crystal structure of yCdc73 will shed new light on the modes of function of Cdc73 and Paf1C.
Acta crystallographica. Section D, Biological crystallography 08/2012; 68(Pt 8):953-9. · 12.67 Impact Factor
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ABSTRACT: Mannonate dehydratase (ManD; EC4.2.1.8) catalyzes the dehydration of d-mannonate to 2-keto-3-deoxygluconate. It is the third enzyme in the pathway for dissimilation of d-glucuronate to 2-keto-3-deoxygluconate involving in the Entner-Doudoroff pathway in certain bacterial and archaeal species. ManD from Gram negative bacteria has an insert sequence as compared to those from Gram positives revealed by sequence analysis. To evaluate the impact of this insert sequence on the catalytic efficiency, we solved the crystal structures of ManD from Escherichia coli strain K12 and its complex with d-mannonate, which reveal that this insert sequence forms two α helices locating above the active site. The two insert α helices introduce a loop that forms a cap covering the substrate binding pocket, which restricts the tunnels of substrate entering and product releasing from the active site. Site-directed mutations and enzymatic activity assays confirm that the catalytic rate is decreased by this loop. These features are conserved among Gram negative bacteria. Thus, the insert sequence of ManD from Gram negative bacteria acts as a common inducer to decrease the catalytic rate and consequently the glucuronate metabolic rate as compared to those from Gram positives. Moreover, residues essential for substrate to enter the active site were characterized via structural analysis and enzymatic activity assays.
Journal of Structural Biology 07/2012; · 3.41 Impact Factor
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ABSTRACT: Palmitoylation/depalmitoylation plays an important role in protein modification. yApt1 is the only enzyme in Saccharomyces cerevisiae that catalyses depalmitoylation. In the present study, recombinant full-length yApt1 was cloned, expressed, purified and crystallized. The crystals diffracted to 2.40 Å resolution and belonged to space group P4(2)2(1)2, with unit-cell parameters a = b = 146.43, c = 93.29 Å. A preliminary model of the three-dimensional structure has been built and further refinement is ongoing.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2012; 68(Pt 7):775-7. · 0.51 Impact Factor
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ABSTRACT: Two cDNAs encoding hemorrhagic snake venom metalloproteinase acutolysin A and non-hemorrhagic metalloproteinase (BR) were
cloned into the expression vector pET-22b, respectively, and the corresponding two recombinant proteins, A-22b and BR-22b,
were produced in inclusion bodies in E. coli BL21(DE3). The recombinant proteins were then subjected to solubilization, purification and refolding in vitro. A-22b showed hemorrhagic activity but no detectable proteolytic activities toward fibrinogen and fibronectin. Natural acutolysin
A had both hemorrhagic activity and proteolytic activity toward these substrates. BR-22b showed the proteolytic activities
toward fibrinogen, but no hemorrhagic activity. In addition, two chimeric genes, C1 and C2, were constructed and cloned into pET-22b, and the corresponding recombinant proteins, C1–22b and C2–22b, were also expressed
in inclusion bodies. C1-22b involved N-terminal 110 amino acids of BR and C-terminal 95 amino acids of acutolysin A, while
C2–22b contained N-terminal 108 amino acids of acutolysin A and C-terminal 112 amino acids of BR. The biological activities
of C2–22b and C1–22b were similar to those of A-22b and BR-22b, respectively. Our results suggested that N-terminal major
subdomain of a snake venom metalloproteinase might play a key role in hemorrhagic activity and have an appreciable effect
on the selectivity for protein substrates.
Chinese Science Bulletin 04/2012; 48(19):2055-2060. · 1.32 Impact Factor
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ABSTRACT: The yeast Shu complex, consisting of the proteins Shu1, Shu2, Psy3, and Csm2, maintains genomic stability by coupling post-replication repair to homologous recombination. However, a lack of biochemical and structural information on the Shu proteins precludes revealing their precise roles within the pathway. Here, we report on the 1.9-Å crystal structure of the Psy3-Csm2 complex. The crystal structure shows that Psy3 forms a heterodimer with Csm2 mainly through a hydrophobic core. Unexpectedly, Psy3 and Csm2 share a similar architecture that closely resembles the ATPase core domain of Rad51. The L2 loop present in Psy3 and Csm2 is similar to that of Rad51 and confers the DNA binding activity of the Shu complex. As with Rad51, the Shu complex appears to form a nucleoprotein filament by binding nonspecifically to DNA. Structure-based mutagenesis studies have demonstrated that the DNA binding activity of the Shu complex is essential for repair of the methyl methanesulfonate-induced DNA damage. Our findings provide good foundations for the understanding of the Srs2 regulation by the Shu complex.
Journal of Biological Chemistry 03/2012; 287(24):20231-9. · 4.77 Impact Factor
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ABSTRACT: Agkisacucetin is a snake C-type lectin isolated from the venom of Agkistrodon acutus (A. acutus). It binds specifically to the platelet glycoprotein (GP) Ib and prevents the von Willebrand factor (VWF) accessing it. We determined the crystal structure of agkisacucetin to 1.9Å resolution. The structure of agkisacucetin has an (αβ) fold similar to another GPIb-binding protein, flavocetin-A, but lacks the C-terminal cysteine in the β-subunit, does not form (βα)(4) tetramers, and does not cluster GPIbs, like flavocetin-A.
Proteins Structure Function and Bioinformatics 02/2012; 80(6):1707-11. · 3.39 Impact Factor
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ABSTRACT: Fanconi anaemia is a rare genetic disease characterized by chromosomal instability and cancer susceptibility. The Fanconi anaemia complementation group protein M (FANCM) forms an evolutionarily conserved DNA-processing complex with MHF1/MHF2 (histone-fold-containing proteins), which is essential for DNA repair in response to genotoxic stress. Here we present the crystal structures of the MHF1-MHF2 complex alone and bound to a fragment of FANCM (FANCM(661-800), designated FANCM-F). The structures show that MHF1 and MHF2 form a compact tetramer to which FANCM-F binds through a 'dual-V' shaped structure. FANCM-F and (MHF1-MHF2)(2) cooperate to constitute a new DNA-binding site that is coupled to the canonical L1L2 region. Perturbation of the MHF-FANCM-F structural plasticity changes the localization of FANCM in vivo. The MHF-FANCM interaction and its subcellular localization are altered by a disease-associated mutant of FANCM. These findings reveal the molecular basis of MHF-FANCM recognition and provide mechanistic insights into the pathway leading to Fanconi anaemia.
Nature Communications 01/2012; 3:782. · 7.40 Impact Factor
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ABSTRACT: Human Pax2 transactivation domain-interacting protein (hPTIP), containing six BRCT domains, is an essential protein required for the IR induced DDR process with an unclear role. Here we report that the tandem BRCT5-BRCT6 domain of hPTIP recognizes the γH2AX tail, and this interaction depends on the phosphorylation of H2AX Ser139 and binding with the carboxyl ending peptide to the aminoacyl ending peptide. The 2.15 Å crystal structure of hPTIP BRCT5/6-γH2AX complex and mutation analysis provide molecular evidence for direct interactions between PTIP and γH2AX. This interaction proffers a new clue to identify the role of PTIP in DDR pathways.
FEBS letters 11/2011; 585(24):3874-9. · 3.54 Impact Factor
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ABSTRACT: Snake-venom thrombin-like enzymes (SVTLEs) are serine proteases that are widely distributed in snakes from the Crotalinae subfamily of the Viperidae. In contrast to other snake-venom serine proteases, they have a biochemical activity similar to that of thrombin and play an important role in the process of blood coagulation. However, SVTLEs cannot activate factor VIII, which is essential in blood-clot stabilization. Consequently, blood clots produced by SVTLEs are not stable and are cleared rapidly. This characteristic makes SVTLEs attractive as potential candidates for antithrombotic therapy. Saxthrombin, an SVTLE from Gloydius saxatilis, was purified and crystallized to obtain a high-quality crystal, from which data were acquired to 1.43 Å resolution. Preliminary X-ray diffraction analysis showed that the crystal belonged to space group C2, with unit-cell parameters a = 94.2, b = 52.2, c = 50.1 Å, β = 96.7°. The crystal structure was determined by molecular replacement and the final R factor was 18.69%; the R(free) was 20.01%. This is the first report of a crystal structure of an SVTLE. Saxthrombin belongs to the typical α/β-hydrolase fold of serine proteases. Its structure was compared with those of thrombin and other snake-venom serine proteases. The observed differences in the amino-acid composition of the loops surrounding the active site appear to contribute to different surface-charge distributions and thus alter the shape of the active-site cleft, which may explain the differences in substrate affinity.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2011; 67(Pt 8):862-5. · 0.51 Impact Factor
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ABSTRACT: SNARE proteins are crucial for membrane fusion in vesicular transport. To ensure efficient and accurate fusion, SNAREs need to be sorted into different budding vesicles. This process is usually regulated by specific recognition between SNAREs and their adaptor proteins. How different pairs of SNAREs and adaptors achieve their recognition is unclear. Here, we report the recognition between yeast SNARE Vti1p and its adaptor Ent3p derived from three crystal structures. Surprisingly, this yeast pair Vti1p/Ent3p interacts through a distinct binding site compared to their homologues vti1b/epsinR in mammals. An opposite surface on Vti1p_Habc domain binds to a conserved area on the epsin N-terminal homology (ENTH) domain of Ent3p. Two-hybrid, in vitro pull-down and in vivo experiments indicate this binding interface is important for correct localization of Vti1p in the cell. This previously undescribed discovery that a cargo and adaptor pair uses different binding sites across species suggests the diversity of SNARE-adaptor recognition in vesicular transport.
Proceedings of the National Academy of Sciences 07/2011; 108(30):12277-82. · 9.68 Impact Factor
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Huihao Zhou,
Zhao Zha,
Yang Liu,
Hongtao Zhang,
Juanjuan Zhu,
Siyi Hu,
Guodong Shen,
Liansheng Cheng, Liwen Niu,
Mark I Greene,
Maikun Teng,
Jing Liu
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ABSTRACT: p185(her2/neu) belongs to the ErbB receptor tyrosine kinase family, which has been associated with human breast, ovarian, and lung cancers. Targeted therapies employing ectodomain-specific p185(her2/neu) monoclonal antibodies (mAbs) have demonstrated clinical efficacy for breast cancer. Our previous studies have shown that p185(her2/neu) mAbs are able to disable the kinase activity of homomeric and heteromeric kinase complexes and induce the conversion of the malignant to normal phenotype. We previously developed a chimeric antibody chA21 that specifically inhibits the growth of p185(her2/neu)-overexpressing cancer cells in vitro and in vivo. Herein, we report the crystal structure of the single-chain Fv of chA21 in complex with an N-terminal fragment of p185(her2/neu), which reveals that chA21 binds a region opposite to the dimerization interface, indicating that chA21 does not directly disrupt the dimerization. In contrast, the bivalent chA21 leads to internalization and down-regulation of p185(her2/neu). We propose a structure-based model in which chA21 cross-links two p185(her2/neu) molecules on separate homo- or heterodimers to form a large oligomer in the cell membrane. This model reveals a mechanism for mAbs to drive the receptors into the internalization/degradation path from the inactive hypophosphorylated tetramers formed dynamically by active dimers during a "physiologic process."
Journal of Biological Chemistry 06/2011; 286(36):31676-83. · 4.77 Impact Factor
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Huihao Zhou,
Zhao Zha,
Yang Liu,
Hongtao Zhang,
Juanjuan Zhu,
Siyi Hu,
Guodong Shen,
Liansheng Cheng, Liwen Niu,
Mark I. Greene,
Maikun Teng,
Jing Liu
[show abstract]
[hide abstract]
ABSTRACT: p185her2/neu belongs to the erbB receptor tyrosine kinase family which has been associated with human breast, ovarian and
lung cancers. Targeted therapies employing ectodomain specific p185her2/neu monoclonal antibodies (mAb) have demonstrated
clinical efficacy for breast cancer. Our previous studies have shown that p185her2/neu mAb are able to disable the kinase
activity of homomeric and heteromeric kinase complexes and induce the conversion of the malignant to normal phenotype. We
previously developed a chimeric antibody chA21 that specifically inhibits the growth of p185her2/neu -over-expressing cancer
cells in vitro and in vivo. Herein, we report the crystal structure of the single-chain Fv of chA21 in complex with an N-terminal
fragment of p185her2/neu, which reveals that chA21 binds a region opposite to the dimerization interface, indicating that
chA21 does not directly disrupt the dimerization. In contrast, the bivalent chA21 leads to internalization and down-regulation
of p185her2/neu. We propose a structure based model in which chA21 cross-links two p185her2/neu molecules on separate homo-
or hetero- dimers to form a large oligomer in the cell membrane. This model reveals a mechanism for mAb to drive the receptors
into the internalization/degradation path from the inactive hypophosphorylated tetramers formed dynamically by active dimers
during a "physiologic process".
Journal of Biological Chemistry 06/2011; · 4.77 Impact Factor
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ABSTRACT: Human antigen R (HuR), a ubiquitously expressed member of the Hu protein family, is an important post-transcriptional regulator which has three RNA-recognition motif (RRM) domains. The two tandem N-terminal RRM domains can selectively bind to the AU-rich element (ARE), while the third one interacts with the poly(A) tail and other proteins. Here, the recombinant ARE-binding region of HuR (residues 18-186) was crystallized in space group P2(1)2(1)2, with unit-cell parameters a = 41.2, b = 133.1, c = 31.4 Å. X-ray diffraction data were collected to a resolution of 2.8 Å. Mutagenesis analysis and SPR assays revealed its poly(U)-binding properties.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 05/2011; 67(Pt 5):546-50. · 0.51 Impact Factor
