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1. chinaXiv:201605.01519 [pdf]

Biochemical and structural characterization of a novel ubiquitin-conjugating enzyme E2 from Agrocybe aegeria reveals Ube2w family-specific properties

Qi, Chao; Li, De-Feng; Feng, Lei; Hou, Yanjie; Wang, Da-Cheng; Qi, Chao; Sun, Hui; Liu, Wei
Subjects: Biology >> Biophysics

Ubiquitination is a post-translational modification that is involved in myriad cellar regulation and disease pathways. The ubiquitin-conjugating enzyme (E2) is an important player in the ubiquitin transfer pathway. Although many E2 structures are available, not all E2 families have known structures, and three-dimensional structures from fungal organisms other than yeast are lacking. We report here the crystal structure of UbcA1, which is a novel ubiquitin-conjugating enzyme identified from the edible and medicinal mushroom Agrocybe aegerita and displays potential antitumor properties. The protein belongs to the Ube2w family and shows similar biochemical characteristics to human Ube2w, including monomer-dimer equilibrium in solution, alpha-NH2 ubiquitin-transfer activity and a mechanism to recognize backbone atoms of intrinsically disordered N-termini in substrates. Its structure displays a unique C-terminal conformation with an orientation of helix alpha 3 that is completely different from the reported E2 structures but similar to a recently reported NMR ensemble of Ube2w. A mutagenesis study on this novel enzyme revealed that an intact C-terminus is significant for protein dimerization and enzymatic activity. As the first crystallized full-length protein of this family, UbcA1 may supersede the truncated X-ray structure of Ube2w (PDB entry 2A7L) as the representative structure of the Ube2w family.

submitted time 2016-05-12 Hits2482Downloads1311 Comment 0

2. chinaXiv:201605.01459 [pdf]

Structure and function of Mycobacterium smegmatis 7-keto-8-aminopelargonic acid (KAPA) synthase

Fan, Shanghua; Chen, Guanjun; Li, De-Feng; Wang, Da-Cheng; Fleming, Joy; Zhang, Hongtai; Zhou, Ying; Zhang, Xian-En; Bi, Lijun; Li, De-Feng; Wang, Da-Cheng; Fleming, Joy; Zhang, Hongtai; Zhou, Ying; Zhang, Xian-En; Bi, Lijun; Zhou, Lin; Chen, Tao; Zhou, Jie
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

The biotin biosynthesis pathway is an attractive target for development of novel drugs against mycobacterial pathogens, however there are as yet no suitable inhibitors that target this pathway in mycobacteria. 7-Keto-8-aminopelargonic acid synthase (KAPA synthase, BioF) is the enzyme which catalyzes the first committed step of the biotin synthesis pathway, but both its structure and function in mycobacteria remain unresolved. Here we present the crystal structure of Mycobacterium smegmatis BioF (MsBioF). The structure reveals an incomplete dimer, and the active site organization is similar to, but distinct from Escherichia coli 8-amino-7-oxononanoate synthase (EcAONS), the E. coli homologue of BioF. To investigate the influence of structural characteristics on the function of MsBioF, we deleted bioF in M. smegmatis and confirmed that BioF is required for growth in the absence of exogenous biotin. Based on structural and mutagenesis studies, we confirmed that pyridoxal 5'-phosphate (PLP) binding site residues His129, Lys235 and His200 are essential for MsBioF activity in vivo and residue Glul 71 plays an important, but not essential role in MsBioF activity. The N-terminus (residues 1-37) is also essential for MsBioF activity in vivo. The structure and function of MsBioF reported here provides further insights for developing new anti-tuberculosis inhibitors aimed at the biotin synthesis pathway. (C) 2014 Elsevier Ltd. All rights reserved.

submitted time 2016-05-12 Hits1707Downloads991 Comment 0

3. chinaXiv:201605.01433 [pdf]

Purification, crystallization and preliminary X-ray crystallographic studies of Rv3899c from Mycobacterium tuberculosis

Song, Yingjia; Li, Honglin; Bi, Lijun; Liu, Jianghui; Wang, Shihua; Liu, Jianghui; Wang, Shihua; Li, De-Feng; Wang, Da-Cheng; Bi, Lijun; Li, De-Feng; Wang, Da-Cheng; Bi, Lijun; Zhou, Jie
Subjects: Biology >> Biophysics

Rv3899c, a hypothetical protein from Mycobacterium tuberculosis that is conserved within the mycobacteria, is predicted to be secreted and has been found in culture filtrates. Here, Rv3899c has been cloned, expressed in Escherichia coli and purified using standard chromatographic techniques. The hanging-drop vapour-diffusion method with PEG 3350 as a precipitant was used to crystallize the protein. N-terminal sequencing results showed that the amino-acid sequence of the crystallized protein began with GATAG, indicating that it is a fragment containing residues 184-410 of Rv3899c. Rv3899c(184-410) crystals exhibited the symmetry of space group P2(1)2(1)2(1), with unit-cell parameters a = 49.88, b = 54.72, c = 75.52 angstrom, = = = 90 degrees, and diffracted to a resolution of 1.90 angstrom.

submitted time 2016-05-12 Hits1508Downloads825 Comment 0

4. chinaXiv:201605.01400 [pdf]

Core component EccB1 of the Mycobacterium tuberculosis type VII secretion system is a periplasmic ATPase

Zhang, Xiao-Li; Zhang, Xian-En; Zhang, Xiao-Li; Li, De-Feng; Fleming, Joy; Wang, Li-Wei; Zhou, Ying; Wang, Da-Cheng; Zhang, Xian-En; Bi, Li-Jun; Zhang, Xiao-Li; Li, De-Feng; Fleming, Joy; Wang, Li-Wei; Zhou, Ying; Wang, Da-Cheng; Zhang, Xian-En; Bi, Li-Jun; Zhang, Xiao-Li
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

Pathogenic mycobacteria transport virulence factors across their complex cell wall via a type VII secretion system (T7SS)/early secreted antigenic target-6 of kDa secretion system (ESX). ESX conserved component (Ecc) B, a core component of the T7SS architecture, is predicted to be a membrane bound protein, but little is known about its structure and function. Here, we characterize EccB1, showing that it is an ATPase with no sequence or structural homology to other ATPases located in the cell envelope of Mycobacterium tuberculosis H37Rv. We obtained the crystal structure of an EccB1-DN72 truncated transmembrane helix and performed modeling and ATP docking studies, showing that EccB1 likely exists as a hexamer. Sequence alignment and ATPase activity determination of EccB1 homologues indicated the presence of 3 conserved motifs in the N- and C-terminals of EccB1-DN72 that assemble together between 2 membrane proximal domains of the EccB1-DN72 monomer. Models of the EccB1 hexamer show that 2 of the conserved motifs are involved in ATPase activity and form an ATP binding pocket located on the surface of 2 adjacent molecules. Our results suggest that EccB may act as the energy provider in the transport of T7SS virulence factors and may be involved in the formation of a channel across the mycomembrane.

submitted time 2016-05-12 Hits2152Downloads1342 Comment 0

5. chinaXiv:201605.01391 [pdf]

Structural Basis of Specific Recognition of Non-Reducing Terminal N-Acetylglucosamine by an Agrocybe aegerita Lectin

Ren, Xiao-Ming; Li, De-Feng; Hu, Yonglin; Wang, Da-Cheng; Ren, Xiao-Ming; Jiang, Shuai; Lan, Xian-Qing; Sun, Hui
Subjects: Biology >> Biophysics

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification that plays essential roles in many cellular pathways. Research in this field, however, is hampered by the lack of suitable probes to identify, accumulate, and purify the O-GlcNAcylated proteins. We have previously reported the identification of a lectin from the mushroom Agrocybe aegerita, i.e., Agrocybe aegerita lectin 2, or AAL2, that could bind terminal N-acetylglucosamine with higher affinities and specificity than other currently used probes. In this paper, we report the crystal structures of AAL2 and its complexes with GlcNAc and GlcNAc beta 1-3Gal beta 1-4GlcNAc and reveal the structural basis of GlcNAc recognition by AAL2 and residues essential for the binding of terminal N-acetylglucosamine. Study on AAL2 may enable us to design a protein probe that can be used to identify and purify O-GlcNAcylated proteins more efficiently.

submitted time 2016-05-12 Hits1557Downloads815 Comment 0

6. chinaXiv:201605.01377 [pdf]

Structure and Self-assembly of NLRP10

Leng Fang-Wei; Wang Da-Cheng; Leng Fang-Wei; Wang Da-Cheng; Leng Fang-Wei; Xie Can
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

NLRP10 is a special member of NOD-like receptors (NLRs) family that lacks the leucine-rich repeats, suggesting that NLRP10 may act as an immunity regulator rather than directly receptor recognizing intracellular pathogen products. Previous studies on NLRP10 show that NLRP10 can interact with several components of NOD1 pathway thereby enhancing NOD1-mediated innate immune responses. In particular models, NLRP10 also negatively affects the activation of NLRP3 inflammasome. It has been proposed that NLRP10 oligomers interact with ASC to form a multi-protein platform for the recruitment of caspase-1 or other signaling components. Here, we show that pyrin-like domain (PYD) degradation induce the formation of NLRP10 oligomers, which present stick-shaped and circular structure. With the NLRP10 mutant G173A made by means of site-directed mutagenesis, we successfully obtain homogeneous full-length NLRP10 preparations. Corresponding gel filtration analysis and electron microscope (EM) data further proved that the PYD domain is important in protecting NLRP10 against aggregation.

submitted time 2016-05-12 Hits1363Downloads683 Comment 0

7. chinaXiv:201605.01244 [pdf]

Structural Insights into the Molecular Recognition between Cerebral Cavernous Malformation 2 and Mitogen-Activated Protein Kinase Kinase Kinase 3

Wang, Xiaoyan; Hou, Yanjie; Deng, Kai; Zhang, Ying; Wang, Da-Cheng; Ding, Jingjin; Wang, Xiaoyan; Deng, Kai
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

Cerebral cavernous malformation 2 (CCM2) functions as an adaptor protein implicated in various biological processes. By interacting with the mitogen-activated protein kinase MEKK3, CCM2 either mediates the activation of MEKK3 signaling in response to osmotic stress or negatively regulates MEKK3 signaling, which is important for normal cardiovascular development. However, the molecular basis governing CCM2-MEKK3 interaction is largely unknown. Here we report the crystal structure of the CCM2 C-terminal part (CCM2ct) containing both the five-helix domain (CCM2ct(s)) and the following C-terminal tail. The end of the C-terminal tail forms an isolated helix, which interacts intramolecularly with CCM2ct(s). By biochemical studies we identified the N-terminal amphiphilic helix of MEKK3 (MEKK3-n(helix)) as the essential structural element for CCM2ct binding. We further determined the crystal structure of CCM2ct(s)-MEKK3-n(helix) complex, in which MEKK3-n(helix) binds to the same site of CCM2ct(s) for CCM2ct intramolecular interaction. These findings build a structural framework for understanding CCM2ct-MEKK3 molecular recognition.

submitted time 2016-05-11 Hits1746Downloads942 Comment 0

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