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ABSTRACT: GNA1162, a predicted lipoprotein from Neisseria meningitidis, is a potential candidate for a universal vaccine against meningococcal disease caused by N. meningitidis serogroup B. Here, the crystal structure of GNA1162 at 1.89 Å resolution determined by single-wavelength anomalous dispersion (SAD) is reported. The structure of GNA1162 appears to be a dimer in the crystallographic asymmetric unit as well as in solution. The overall structure of the dimer indicates that each monomer inserts its C-terminal α5 helix into the hydrophobic groove of the other molecule. Moreover, the β4 strands of each monomer lie antiparallel to each other and interact through multiple main-chain hydrogen bonds. Through structural comparisons and operon predictions, it is hypothesized that GNA1162 is part of a transport system and assists in transport and reassembly. The crystal structure of GNA1162 sheds light on its possible function and provides potentially valuable information for the design of a vaccine against meningococcal disease.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 04/2013; 69(Pt 4):362-8. · 0.51 Impact Factor
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ABSTRACT: SAD-1 is a serine/threonine kinase which plays an important role in the regulation of both neuronal polarity and synapse formation in Caenorhabditis elegans. The kinase domain of SAD-1 from C. elegans was overexpressed in Escherichia coli BL21 (DE3) cells and purified to homogeneity using nickel-nitrilotriacetic acid metal-affinity, ion-exchange and gel-filtration chromatography. Diffraction-quality crystals were grown using the sitting-drop vapour-diffusion technique from a condition consisting of 1 M CAPSO pH 9.6, 10%(w/v) polyethylene glycol 3350. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 205.4, b = 57.1, c = 71.7 Å, β = 106.1°. X-ray diffraction data were recorded to 3.0 Å resolution from a single crystal using synchrotron radiation.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 04/2013; 69(Pt 4):449-52. · 0.51 Impact Factor
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ABSTRACT: The amyloid-β protein precursor (APP) plays a crucial role in the pathogenesis of Alzheimer's disease (AD). Knock-out and transgenic mouse studies of the adaptor protein Mint2 have revealed that it is a major player in regulating APP metabolism physiologically through the binding of its phosphotyrosine-binding (PTB) domain to the intracellular domain of APP. However, the molecular mechanism of APP dynamically binding to Mint2 remains elusive. Here, we report the structures of APP peptide-free and APP peptide-bound C-terminal Mint2 mutants at resolutions of 2.7 and 3.3 Å, respectively. Our structures reveal that APP peptide-free Mint2 exists in a closed state in which the ARM domain blocks the peptide-binding groove of the PTB domain. In sharp contrast, APP peptide-bound Mint2 exists in an open state in which the ARM domain drastically swings away from the bound peptide. Mutants that control the open-closed motion of Mint2 dynamically regulated APP metabolism both in vitro and in vivo. Our results uncover a novel open-closed mechanism of the PTB domain dynamically binding to its peptide substrate. Moreover, such a conformational switch may represent a general regulation mode of APP family members by Mint proteins, providing useful information for the treatment of AD.
Journal of Molecular Cell Biology 06/2012;
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ABSTRACT: Elongator is a multiprotein complex composed of two subcomplexes, Elp1-3 and Elp4-6. Elongator is highly conserved between yeast and humans and plays an important role in RNA polymerase II-mediated transcriptional elongation and many other processes, including cytoskeleton organization, exocytosis, and tRNA modification. Here, we determined the crystal structure of the Elp4-6 subcomplex of yeast. The overall structure of Elp4-6 revealed that Elp6 acts as a bridge to assemble Elp4 and Elp5. Detailed structural and sequence analyses revealed that each subunit in the Elp4-6 subcomplex forms a RecA-ATPase-like fold, although it lacks the key sequence signature of ATPases. Site-directed mutagenesis and biochemical analyses indicated that the Elp4-6 subcomplex can assemble into a hexameric ring-shaped structure in vitro and in vivo. Furthermore, GST pulldown assays showed that the ring-shaped assembly of the Elp4-6 subcomplex is important for its specific histone H3 binding. Our results may shed light on the substrate recognition and assembly of the holo-Elongator complex.
Journal of Biological Chemistry 05/2012; 287(25):21501-8. · 4.77 Impact Factor
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ABSTRACT: Calcium influx through the Ca(2+) release-activated Ca(2+) (CRAC) channel is an essential process in many types of cells. Upon store depletion, the calcium sensor in the endoplasmic reticulum, STIM1, activates Orai1, a CRAC channel in the plasma membrane. We have determined the structures of SOAR from Homo sapiens (hSOAR), which is part of STIM1 and is capable of constitutively activating Orai1, and the entire coiled coil region of STIM1 from Caenorhabditis elegans (ceSTIM1-CCR) in an inactive state. Our studies reveal that the formation of a SOAR dimer is necessary to activate the Orai1 channel. Mutations that disrupt SOAR dimerization or remove the cluster of positive residues abolish STIM1 activation of Orai1. We identified a possible inhibitory helix within the structure of ceSTIM1-CCR that tightly interacts with SOAR. Functional studies suggest that the inhibitory helix may keep the C-terminus of STIM1 in an inactive state. Our data allowed us to propose a model for STIM1 activation.
Proceedings of the National Academy of Sciences 03/2012; 109(15):5657-62. · 9.68 Impact Factor
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ABSTRACT: The assembly of supramolecular complexes in multidomain scaffold proteins is crucial for the control of cell polarity. The scaffold protein of protein associated with Lin-7 1 (Pals1) forms a complex with two other scaffold proteins, Pals-associated tight junction protein (Patj) and mammalian homolog-2 of Lin-7 (Mals2), through its tandem Lin-2 and Lin-7 (L27) domains to regulate apical-basal polarity. Here, we report the crystal structure of a 4-L27 domain-containing heterotrimer derived from the tripartite complex Patj/Pals1/Mals2. The heterotrimer consists of two cognate pairs of heterodimeric L27 domains with similar conformations. Structural analysis and biochemical data further show that the dimers assemble mutually independently. Additionally, such mutually independent assembly of the two heterodimers can be observed in another tripartite complex, Disks large homolog 1 (DLG1)/calcium-calmodulin-dependent serine protein kinase (CASK)/Mals2. Our results reveal a novel mechanism for tandem L27 domain-mediated, supramolecular complex assembly with a mutually independent mode.
Journal of Biological Chemistry 02/2012; 287(14):11132-40. · 4.77 Impact Factor
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ABSTRACT: Special AT-rich sequence-binding protein 1 (SATB1) is a global chromatin organizer and gene expression regulator essential for T-cell development and breast cancer tumor growth and metastasis. The oligomerization of the N-terminal domain of SATB1 is critical for its biological function. We determined the crystal structure of the N-terminal domain of SATB1. Surprisingly, this domain resembles a ubiquitin domain instead of the previously proposed PDZ domain. Our results also reveal that SATB1 can form a tetramer through its N-terminal domain. The tetramerization of SATB1 plays an essential role in its binding to highly specialized DNA sequences. Furthermore, isothermal titration calorimetry results indicate that the SATB1 tetramer can bind simultaneously to two DNA targets. Based on these results, we propose a molecular model whereby SATB1 regulates the expression of multiple genes both locally and at a distance.
Nucleic Acids Research 01/2012; 40(9):4193-202. · 8.03 Impact Factor
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ABSTRACT: MyD88 adaptor-like protein (Mal) is a crucial adaptor that acts as a bridge to recruit the MyD88 molecule to activated TLR4 receptors in response to invading pathogens. The specific assembly of the Toll/interleukin-1 receptor (TIR) domains of TLR4, Mal and MyD88 is responsible for proper signal transduction in the TLR4 signaling pathway. However, the molecular mechanism for the specificity of these TIR domains remains unclear. Here, we present the crystal structure of the TIR domain of the human Mal molecule (Mal-TIR) at a resolution of 2.4 Å. Unexpectedly, Mal-TIR exhibits an extraordinarily long AB loop, but no αB helix or BB loop, distinguishing it from other TIR domains. More importantly, the Mal-TIR AB loop is capable of mediating direct binding to the TIR domains of TLR4 and MyD88 simultaneously. We also found that Mal-TIR can form a back-to-back dimer that may resemble the dimeric assembly of the entire Mal molecule. Our data demonstrate the bridge role of the Mal-TIR domain and provide important information about TIR domain specificity.
PLoS ONE 01/2012; 7(4):e34202. · 4.09 Impact Factor
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ABSTRACT: The CBM complex (CARMA1, BCL10 and MALT1) plays a crucial role in B and T lymphocyte activation. CARMA1 serves as a scaffold for BCL10, MALT1 and other effector proteins and regulates various signaling pathways related to the immune response. The assembly of CARMA1 and BCL10 is mediated through a CARD-CARD interaction. Here, we report the crystal structure of the CARD domain of CARMA1 at a resolution of 1.75 Å. The structure consists of six helices, as previously determined for CARD domains. Structural and computational analysis identified the binding interface between CARMA1-CARD and BCL10-CARD, which consists of a basic patch in CARMA1 and an acidic patch in BCL10. Site-directed mutagenesis, co-immunoprecipitation and an NF-κB activation assay confirmed that the interface is necessary for association and downstream signaling. Our studies provide molecular insight into the assembly of CARMA1 and BCL10.
PLoS ONE 01/2012; 7(8):e42775. · 4.09 Impact Factor
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ABSTRACT: The L27 (LIN-2/LIN-7) domain is a protein-protein interaction module capable of assembling proteins into biologically important complexes. Pals1 contains two L27 domains: the first, L27N, interacts with PATJ, and the second, L27C, interacts with MALS, forming a tripartite complex that plays a crucial role in the establishment and maintenance of cell polarity. To provide a better understanding of the mechanism of assembly of this tripartite complex, four different L27(PATJ)-(L27N,L27C)(Pals1)-L27(MALS) constructs were cloned, expressed, purified and crystallized. Crystals of tripartite complex 1 of L27(PATJ)-(L27N,L27C)(Pals1)-L27(MALS) diffracted to 2.05 Å resolution. These crystals belonged to either space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 145.2, c = 202.5 Å. Assuming the presence of four molecules in the asymmetric unit, a Matthews coefficient of 2.69 Å(3) Da(-1) was calculated, corresponding to a solvent content of 54.25%.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 11/2011; 67(Pt 11):1443-7. · 0.51 Impact Factor
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ABSTRACT: Human maltase-glucoamylase (MGAM) hydrolyzes linear alpha-1,4-linked oligosaccharide substrates, playing a crucial role in the production of glucose in the human lumen and acting as an efficient drug target for type 2 diabetes and obesity. The amino- and carboxyl-terminal portions of MGAM (MGAM-N and MGAM-C) carry out the same catalytic reaction but have different substrate specificities. In this study, we report crystal structures of MGAM-C alone at a resolution of 3.1 Å, and in complex with its inhibitor acarbose at a resolution of 2.9 Å. Structural studies, combined with biochemical analysis, revealed that a segment of 21 amino acids in the active site of MGAM-C forms additional sugar subsites (+ 2 and + 3 subsites), accounting for the preference for longer substrates of MAGM-C compared with that of MGAM-N. Moreover, we discovered that a single mutation of Trp1251 to tyrosine in MGAM-C imparts a novel catalytic ability to digest branched alpha-1,6-linked oligosaccharides. These results provide important information for understanding the substrate specificity of alpha-glucosidases during the process of terminal starch digestion, and for designing more efficient drugs to control type 2 diabetes or obesity.
Protein & Cell 10/2011; 2(10):827-36.
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ABSTRACT: NMB0315 is an outer membrane protein of Neisseria meningitidis serogroup B (NMB) and a potential candidate for a broad-spectrum vaccine against meningococcal disease. The crystal structure of NMB0315 was solved by single-wavelength anomalous dispersion (SAD) at a resolution of 2.4 Å and revealed to be a lysostaphin-type peptidase of the M23 metallopeptidase family. The overall structure consists of three well-separated domains and has no similarity to any previously published structure. However, only the topology of the carboxyl-terminal domain is highly conserved among members of this family, and this domain is a zinc-dependent catalytic unit. The amino-terminal domain of the structure blocks the substrate binding pocket in the carboxyl-terminal domain, indicating that the wild-type full-length protein is in an inactive conformational state. Our studies improve the understanding of the catalytic mechanism of M23 metallopeptidases.
PLoS ONE 01/2011; 6(10):e26845. · 4.09 Impact Factor
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Xue Yang,
Xingqiao Xie,
Liu Chen,
Hao Zhou,
Zheng Wang,
Weijing Zhao,
Ran Tian,
Rongguang Zhang,
Changlin Tian,
Jiafu Long, Yuequan Shen
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ABSTRACT: The establishment of epithelial cell polarity requires the assembly of multiprotein complexes and is crucial during epithelial morphogenesis. Three scaffolding proteins, Dlg1, MPP7, and Mals3, can be assembled to form a complex that functions in the establishment and maintenance of apicobasal polarity in epithelial tissues through their L27 domains. Here we report the crystal structure of a 4-L27-domain complex derived from the human tripartite complex Dlg1-MPP7-Mals3 in combination with paramagnetic relaxation enhancement measurements. The heterotrimer consists of 2 pairs of heterodimeric L27 domains. These 2 dimers are asymmetric due to the large difference between the N- and C-terminal tandem L27 domain of MPP7. Structural analysis combined with biochemical experiments further reveals that the loop αA-αB and helix αB of the C-terminal L27 domain of MPP7 play a critical role in assembling the entire tripartite complex, suggesting a synergistic tandem L27-mediated assembling event.
The FASEB Journal 12/2010; 24(12):4806-15. · 5.71 Impact Factor
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ABSTRACT: Human protoporphyrinogen IX oxidase (hPPO), a mitochondrial inner membrane protein, converts protoporphyrinogen IX to protoporphyrin IX in the heme biosynthetic pathway. Mutations in the hPPO gene cause the inherited human disease variegate porphyria (VP). In this study, we report the crystal structure of hPPO in complex with the coenzyme flavin adenine dinucleotide (FAD) and the inhibitor acifluorfen at a resolution of 1.9 Å. The structural and biochemical analyses revealed the molecular details of FAD and acifluorfen binding to hPPO as well as the interactions of the substrate with hPPO. Structural analysis and gel chromatography indicated that hPPO is a monomer rather than a homodimer in vitro. The founder-effect mutation R59W in VP patients is most likely caused by a severe electrostatic hindrance in the hydrophilic binding pocket involving the bulky, hydrophobic indolyl ring of the tryptophan. Forty-seven VP-causing mutations were purified by chromatography and kinetically characterized in vitro. The effect of each mutation was demonstrated in the high-resolution crystal structure.
The FASEB Journal 11/2010; 25(2):653-64. · 5.71 Impact Factor
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ABSTRACT: Human pancreatic α-amylase (HPA) catalyzes the hydrolysis of α-d-(1,4) glycosidic linkages in starch and is one of the major therapeutic targets for type II diabetes. Several acarviostatins isolated from Streptomyces coelicoflavus var. nankaiensis previously showed more potent inhibition of HPA than acarbose, which has been successfully used in clinical therapy. However, the molecular mechanisms by which acarviostatins inhibit HPA remains elusive. Here we determined crystal structures of HPA in complexes with a series of acarviostatin inhibitors (I03, II03, III03, and IV03). Structural analyses showed that acarviostatin I03 undergoes a series of hydrolysis and condensation reactions in the HPA active site, similar to acarbose, while acarviostatins II03, III03, and IV03 likely undergo only hydrolysis reactions. On the basis of structural analysis combined with kinetic assays, we demonstrate that the final modified product with seven sugar rings is best suited for occupying the full active site and shows the most efficient inhibition of HPA. Our high resolution structures reported here identify first time an interaction between an inhibitor and subsite-4 of the HPA active site, which we show makes a significant contribution to the inhibitory effect. Our results provide important information for the design of new drugs for the treatment of type II diabetes or obesity.
Journal of Structural Biology 11/2010; 174(1):196-202. · 3.41 Impact Factor
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ABSTRACT: Integrase plays a critical role in the recombination of viral DNA into the host genome. Therefore, over the past decade, it has been a hot target of drug design in the fight against type 1 human immunodeficiency virus (HIV-1). Bovine immunodeficiency virus (BIV) integrase has the same function as HIV-1 integrase. We have determined crystal structures of the BIV integrase catalytic core domain (CCD) in two different crystal forms at a resolution of 2.45 Å and 2.2 Å, respectively. In crystal form I, BIV integrase CCD forms a back-to-back dimer, in which the two active sites are on opposite sides. This has also been seen in many of the CCD structures of HIV-1 integrase that were determined previously. However, in crystal form II, BIV integrase CCD forms a novel face-to-face dimer in which the two active sites are close to each other. Strikingly, the distance separating the two active sites is approximately 20 Å, a distance that perfectly matches a 5-base pair interval. Based on these data, we propose a model for the interaction of integrase with its target DNA, which is also supported by many published biochemical data. Our results provide important clues for designing new inhibitors against HIV-1.
Protein & Cell 04/2010; 1(4):363-70.
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ABSTRACT: Protoporphyrinogen IX oxidase (PPO) converts protoporphyrinogen IX to protoporphyrin IX, playing an important part in the heme/chlorophyll biosynthetic pathway. Bacillus subtilis PPO (bsPPO) is unique among PPO family members in that it is a soluble monomer, is inefficiently inhibited by the herbicide acifluorfen (AF) and has broader substrate specificity than other PPO enzymes. Here, we present the crystal structure of bsPPO bound to AF. Our structure shows that the AF molecule binds to a new site outside the previously identified inhibitor binding pocket. Most importantly, the benzene ring of the 2-nitrobenzoic acid moiety of AF lies parallel to the isoalloxazine ring of FAD at a distance of less than 3.5A, providing a framework for the interaction of FAD with the substrate protoporphyrinogen IX. Furthermore, our structure reveals that the larger substrate binding chamber and predominantly positively charged chamber surface of bsPPO are more favorable for the binding of coproporphyrinogen-III. These crystallographic findings uncover biochemically unique properties of bsPPO, providing important information for further understanding the enzymatic mechanism.
Journal of Structural Biology 11/2009; 170(1):76-82. · 3.41 Impact Factor
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ABSTRACT: GNA1946, a conserved outer membrane lipoprotein from Neisseria meningitidis, has been identified as a candidate antigen for an urgently needed broad-spectrum meningococcal vaccine. It has been predicted to be a periplasmic receptor in the D-methionine uptake ABC transporter system. The crystal structure of GNA1946 was solved by the single-wavelength anomalous dispersion (SAD) method to a resolution of 2.25 A, and it reveals a Venus flytrap-like structure. GNA1946 consists of two globular lobes connected by a hinge region. Surprisingly, the structure showed an L-methionine bound within the cleft between the lobes. A comparison of GNA1946 with two other outer membrane lipoproteins, the L-methionine-binding Tp32 from Treponema pallidum and the dipeptide GlyMet-binding protein Pg110 from Staphylococcus aureus, revealed that although these three proteins share low sequence similarities, there is a high degree of structural conservation and similar substrate-binding frameworks. Our results reveal that GNA1946 is an L-methionine binding lipoprotein in the outer membrane, and should function as an initial receptor for ABC transporters with high affinity and specificity. The GNA1946 structure reported here should provide a valuable starting point for the development of a broad-spectrum meningococcal vaccine.
Journal of Structural Biology 10/2009; 168(3):437-43. · 3.41 Impact Factor
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ABSTRACT: Inwardly rectifying potassium channel 2.3 (Kir2.3) is specifically targeted on the basolateral membranes of epithelial and neuronal cells, and it thus plays an important role in maintaining potassium homeostasis. Tax-interacting protein-1 (TIP-1), an atypical PDZ-domain-containing protein, binds to Kir2.3 with a high affinity, causing the intracellular accumulation of Kir2.3 in cultured epithelial cells. However, the molecular basis of the TIP-1/Kir2.3 interaction is still poorly understood. Here, we present the crystal structure of TIP-1 in complex with the C-terminal Kir2.3-peptide (residues 436-445) to reveal the molecular details of the interaction between them. Moreover, isothermal titration calorimetry experiments show that the C-terminal Kir2.3-peptide binds much more strongly to TIP-1 than to mammalian Lin-7, indicating that TIP-1 can compete with mammalian Lin-7 to uncouple Kir2.3 from its basolateral membrane anchoring complex. We further show that the phosphorylation/dephosphorylation of Ser443 within the C-terminal Kir2.3 PDZ-binding motif RRESAI dynamically regulates the Kir2.3/TIP-1 association in heterologous HEK293T cells. These data suggest that TIP-1 may act as an important regulator for the endocytic pathway of Kir2.3.
Journal of Molecular Biology 08/2009; 392(4):967-76. · 4.00 Impact Factor
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ABSTRACT: Protein kinase CK2 consists of two catalytic subunits (CK2α) and two regulatory subunits (CK2β). Here, we report the crystal
structures of rat CK2α mutant (rCK2α-ΔC, 1–335) and CK2β (rCK2β). The overall topology of rCK2α-ΔC and rCK2β are very similar
to the human enzyme, although large structural differences could be observed in the N-terminal domain of rCK2α-ΔC. Our reported
structure of rCK2α-ΔC is in the close conformation state while the counterpart hCK2α is in the open conformation state, indicating
the conformation of CK2α molecule has high plasticity. The structure of rCK2β represents the conformation of free CK2β. Upon
CK2α binding, the C-terminal region undergoes a drastic conformational change. The major region of interaction within the
interface of CK2α/CK2β may serve as a bridge to transmit the conformational change and thus regulate the activity of CK2α.
Chinese Science Bulletin 12/2008; 54(2):220-226. · 1.32 Impact Factor
