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Abstract: NGFI-B is a ligand-independent orphan nuclear receptor of the NR4A subfamily that displays important functional differences with its homolog Nurr1. In particular, the NGFI-B ligand-binding domain (LBD) exhibits only modest activity in cell lines in which the Nurr1 LBD strongly activates transcription. To gain insight into the structural basis for the distinct activation potentials, we determined the crystal structure of the NGFI-B LBD at 2.4-Å resolution. Superimposition with the Nurr1 LBD revealed a significant shift of the position of helix 12, potentially caused by conservative amino acids exchanges in helix 3 or helix 12. Replacement of the helix 11–12 region of Nurr1 with that of NGFI-B dramatically reduces the transcriptional activity of the Nurr1 LBD. Similarly, mutation of Met414 in helix 3 to leucine or of Leu591 in helix 12 to isoleucine (the corresponding residues found in NGFI-B) significantly affects Nurr1 transactivation. In comparison, swapping the helix 11–12 region of Nurr1 into NGFI-B results in a modest increase of activity. These observations reveal a high sensitivity of LBD activity to changes that influence helix 12 positioning. Furthermore, mutation of hydrophobic surface residues in the helix 11–12 region (outside the canonical co-activator surface constituted by helices 3, 4, and 12) severely affects Nurr1 transactivation. Together, our data suggest that a novel co-regulator surface that includes helix 11 and a specifically positioned helix 12 determine the cell type-dependent activities of the NGFI-B and the Nurr1 LBD.
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Feb 2005
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Abstract: Phospho-CDK2/cyclin A, a kinase that is active in cell cycle S phase, contains an RXL substrate recognition site that is over 40 Å from the catalytic site. The role of this recruitment site, which enhances substrate affinity and catalytic efficiency, has been investigated using peptides derived from the natural substrates, namely CDC6 and p107, and a bispeptide inhibitor in which the ?-phosphate of ATP is covalently attached by a linker to the CDC6 substrate peptide. X-ray studies with a 30-residue CDC6 peptide in complex with pCDK2/cyclin A showed binding of a dodecamer peptide at the recruitment site and a heptapeptide at the catalytic site, but no density for the linking 11 residues. Kinetic studies established that the CDC6 peptide had an 18-fold lower Km compared with heptapeptide substrate and that this effect required the recruitment peptide to be covalently linked to the substrate peptide. X-ray studies with the CDC6 bispeptide showed binding of the dodecamer at the recruitment site and the modified ATP in two alternative conformations at the catalytic site. The CDC6 bispeptide was a potent inhibitor competitive with both ATP and peptide substrate of pCDK2/cyclin A activity against a heptapeptide substrate (Ki = 0.83 nm) but less effective against RXL-containing substrates. We discuss how localization at the recruitment site (KD 0.4 ?m) leads to increased catalytic efficiency and the design of a potent inhibitor. The notion of a flexible linker between the sites, which must have more than a minimal number of residues, provides an explanation for recognition and discrimination against different substrates.
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May 2006
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NONE-No attached Diamond beamline
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Misaki
Yamamoto
,
Satoru
Unzai
,
Shinya
Saijo
,
Kazuki
Ito
,
Kenji
Mizutani
,
Chiyo
Suno-ikeda
,
Yukako
Yabuki-miyata
,
Takaho
Terada
,
Mitsutoshi
Toyama
,
Mikako
Shirouzu
,
Takuya
Kobayashi
,
Yoshimi
Kakinuma
,
Ichiro
Yamato
,
Shigeyuki
Yokoyama
,
Takeshi
Murata
,
So
Iwata
Abstract: The vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain and an integral membrane domain connected by a central stalk and a few peripheral stalks. The number and arrangement of the peripheral stalk subunits remain controversial. The peripheral stalk of Na+-translocating V-ATPase from Enterococcus hirae is likely to be composed of NtpE and NtpF (corresponding to subunit G of eukaryotic V-ATPase) subunits together with the N-terminal hydrophilic domain of NtpI (corresponding to subunit a of eukaryotic V-ATPase). Here we purified NtpE, NtpF, and the N-terminal hydrophilic domain of NtpI (NtpINterm) as separate recombinant His-tagged proteins and examined interactions between these three subunits by pulldown assay using one tagged subunit, CD spectroscopy, surface plasmon resonance, and analytical ultracentrifugation. NtpINterm directly bound NtpF, but not NtpE. NtpE bound NtpF tightly. NtpINterm bound the NtpE-F complex stronger than NtpF only, suggesting that NtpE increases the binding affinity between NtpINterm and NtpF. Purified NtpE-F-INterm complex appeared to be monodisperse, and the molecular masses estimated from analytical ultracentrifugation and small-angle x-ray scattering (SAXS) indicated that the ternary complex is formed with a 1:1:1 stoichiometry. A low resolution structure model of the complex produced from the SAXS data showed an elongated “L” shape.
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May 2008
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NONE-No attached Diamond beamline
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Holger
Greschik
,
Magnus
Althage
,
Ralf
Flaig
,
Yoshiteru
Sato
,
Virginie
Chavant
,
Carole
Peluso-iltis
,
Laurence
Choulier
,
Philippe
Cronet
,
Natacha
Rochel
,
Roland
Schüle
,
Per-erik
P.e. Strömstedt
,
Dino
Moras
Abstract: Although structural studies on the ligand-binding domain (LBD) have established the general mode of nuclear receptor (NR)/coactivator interaction, determinants of binding specificity are only partially understood. The LBD of estrogen receptor-? (ER?), for example, interacts only with a region of peroxisome proliferator-activated receptor coactivator (PGC)-1?, which contains the canonical LXXLL motif (NR box2), whereas the LBD of estrogen-related receptor-? (ERR?) also binds efficiently an untypical, LXXYL-containing region (NR box3) of PGC-1?. Surprisingly, in a previous structural study, the ER? LBD has been observed to bind NR box3 of transcriptional intermediary factor (TIF)-2 untypically via LXXYL, whereas the ERR? LBD binds this region of TIF-2 only poorly. Here we present a new crystal structure of the ERR? LBD in complex with a PGC-1? box3 peptide. In this structure, residues N-terminal of the PGC-1? LXXYL motif formed contacts with helix 4, the loop connecting helices 8 and 9, and with the C terminus of the ERR? LBD. Interaction studies using wild-type and mutant PGC-1? and ERR? showed that these contacts are functionally relevant and are required for efficient ERR?/PGC-1? interaction. Furthermore, a structure comparison between ERR? and ER? and mutation analyses provided evidence that the helix 8–9 loop, which differs significantly in both nuclear receptors, is a major determinant of coactivator binding specificity. Finally, our results revealed that in ERR? the helix 8–9 loop allosterically links the LBD homodimer interface with the coactivator cleft, thus providing a plausible explanation for distinct PGC-1? binding to ERR? monomers and homodimers.
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Jul 2008
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I04-Macromolecular Crystallography
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Open Access
Abstract: Adhesive PfEMP1 proteins are displayed on the surface of malaria-infected red blood cells. They play a critical role in the disease, tethering infected cells away from destruction by the spleen and causing many severe symptoms. A molecular understanding of how these domains maintain their binding properties while evading immune detection will be important in developing therapeutics. In malaria of pregnancy, domains from the var2csa-encoded PfEMP1 protein interact with chondroitin sulfate on the placenta surface. This causes accumulation of infected red blood cells, leading to placental inflammation and block of blood flow to the developing fetus. This is associated with maternal anemia, low birth weight, and premature delivery and can lead to the death of mother and child. Here I present the structure of the chondroitin sulfate-binding DBL3X domain from a var2csa-encoded PfEMP1 protein. The domain adopts a fold similar to malarial invasion proteins, with extensive loop insertions. One loop is flexible in the unliganded structure but observed in the presence of sulfate or disaccharide, where it completes a sulfate-binding site. This loop, and others surrounding this putative carbohydrate-binding site, are flexible and polymorphic, perhaps protecting the binding site from immune detection. This suggests a model for how the domain maintains ligand binding while evading the immune response and will guide future drug and vaccine development.
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Aug 2008
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I03-Macromolecular Crystallography
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Open Access
Abstract: Chromosomal instability can result from defective control of checkpoints and is associated with malignant cell growth. Monopolar spindle 1 (Mps1) is a dual-specificity protein kinase that has important roles in the prevention of aneuploidy during the cell cycle and might therefore be a potential target for new therapeutic agents in the treatment of cancer. To gain insights into the molecular mechanism of Mps1 inhibition by small molecules, we determined the x-ray structure of Mps1, both alone and in complex with the ATP-competitive inhibitor SP600125. Mps1 adopts a classic protein kinase fold, with the inhibitor sitting in the ATP-binding site where it is stabilized by hydrophobic interactions. We identified a secondary pocket, not utilized by SP600125, which might be exploited for the rational design of specific Mps1 inhibitors. These structures provide important insights into the interaction of this protein kinase with small molecules and suggest potential mechanisms for Mps1 regulation.
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Aug 2008
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I04-Macromolecular Crystallography
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Abstract: A 2-His-1-carboxylate triad of iron binding residues is present in many non-heme iron oxygenases including the Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenases. Three variants (D201A, D201E, and D201G) of the iron binding Asp-201 residue of an asparaginyl hydroxylase, factor inhibiting HIF (FIH), were made and analyzed. FIH-D201A and FIH-D201E did not catalyze asparaginyl hydroxylation, but in the presence of a reducing agent, they displayed enhanced 2OG turnover when compared with wild-type FIH. Turnover of 2OG by FIH-D201A was significantly stimulated by the addition of HIF-1?786–826 peptide. Like FIH-D201A and D201E, the D201G variant enhanced 2OG turnover but rather unexpectedly catalyzed asparaginyl hydroxylation. Crystal structures of the FIH-D201A and D201G variants in complex with Fe(II)/Zn(II), 2OG, and HIF-1?786–826/788–806 implied that only two FIH-based residues (His-199 and His-279) are required for metal binding. The results indicate that variation of 2OG-dependent dioxygenase iron-ligating residues as a means of functional assignment should be treated with caution. The results are of mechanistic interest in the light of recent biochemical and structural analyses of non-heme iron and 2OG-dependent halogenases that are similar to the FIH-D201A/G variants in that they use only two His-residues to ligate iron.
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Sep 2008
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NONE-No attached Diamond beamline
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Abstract: Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a multi-functioning protein belonging to the lipocalin family, acting as a PGD2-synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. In the present study, to clarify the conformational changes of lipocalin proteins induced by binding of lipophilic ligands, such as all-trans-retinoic acid (RA), bilirubin (BR) and biliverdin (BV), we measured small-angle X-ray scattering (SAXS) of L-PGDS and that of two other lipocalins, ?-lactoglobulin (?LG) and retinol-binding protein (RBP). L-PGDS bound all three ligands with high affinity, while ?LG and RBP could bind only RA. The radius of gyration was estimated to be 19.4 Å for L-PGDS, and 18.8 Å for L-PGDS/RA, 17.3 Å for L-PGDS/BR and 17.8 Å for L-PGDS/BV complexes, indicating that L-PGDS became compact after binding of these ligands. Alternatively, the radius of gyration of ?LG and RBP was 20.3 and 26.2 Å, respectively, and was almost the same before and after RA binding. Based on the SAXS data, we found that the compact packing upon binding ligands is a special feature of L-PGDS and it may be ascribed to the conformational flexibility of L-PGDS molecule itself, which underlies the high-affinity for its ligands.
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Oct 2008
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I03-Macromolecular Crystallography
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Abstract: Sortases are a family of Gram-positive bacterial transpeptidases that anchor secreted proteins to bacterial cell surfaces. These include many proteins that play critical roles in the virulence of Gram-positive bacterial pathogens such that sortases are attractive targets for development of novel antimicrobial agents. All Gram-positive pathogens express a “housekeeping” sortase that recognizes the majority of secreted proteins containing an LPXTG wall-sorting motif and covalently attaches these to bacterial cell wall peptidoglycan. Many Gram-positive pathogens also express additional sortases that link a small number of proteins, often with variant wall-sorting motifs, to either other surface proteins or peptidoglycan. To better understand the mechanisms of catalysis and substrate recognition by the housekeeping sortase produced by the important human pathogen Streptococcus pyogenes, the crystal structure of this protein has been solved and its transpeptidase activity established in vitro. The structure reveals a novel arrangement of key catalytic residues in the active site of a sortase, the first that is consistent with kinetic analysis. The structure also provides a complete description of residue positions surrounding the active site, overcoming the limitation of localized disorder in previous structures of sortase A-type proteins. Modification of the active site Cys through oxidation to its sulfenic acid form or by an alkylating reagent supports a role for a reactive thiol/thiolate in the catalytic mechanism. These new insights into sortase structure and function could have important consequences for inhibitor design.
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Jan 2009
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Abstract: Disruption of Golgi alpha-mannosidase II activity can result in type II congenital dyserythropoietic anemia and induce lupus-like autoimmunity in mice. Here, we isolated a mutant human embryonic kidney (HEK) 293T cell line called Lec36, which displays sensitivity to ricin that lies between the parental HEK 293T cells, in which the secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which produce only oligomannose-type N-linked glycans. Stem cell marker 19A was transiently expressed in the HEK 293T Lec36 cells and in parental HEK 293T cells with and without the potent Golgi alpha-mannosidase II inhibitor, swainsonine. Negative ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, were dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi alpha-mannosidase II: a point mutation that mapped to the active site was found in one allele, and an in-frame deletion of 12 nucleotides was found in the other allele. Expression of the wild type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and as a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia.
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Jan 2009
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