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Schreiter Lab / Publications
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17 Publications

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    10/01/13 | Common genetic variation at the IL1RL1 locus regulates IL-33/ST2 signaling.
    Ho JE, Chen W, Chen M, Larson MG, McCabe EL, Cheng S, Ghorbani A, Coglianese E, Emilsson V, Johnson AD, Walter S, Franceschini N, O'Donnell CJ, CARDIoGRAM Consortium , CHARGE Inflammation Working Group , Dehghan A, Lu C, Levy D, Newton-Cheh C, CHARGE Heart Failure Working Group , Lin H, Felix JF, Schreiter ER, Vasan RS, Januzzi JL, Lee RT, Wang TJ
    The Journal of Clinical Investigation. 2013 Oct;123(10):4208-18. doi: 10.1172/JCI67119

    The suppression of tumorigenicity 2/IL-33 (ST2/IL-33) pathway has been implicated in several immune and inflammatory diseases. ST2 is produced as 2 isoforms. The membrane-bound isoform (ST2L) induces an immune response when bound to its ligand, IL-33. The other isoform is a soluble protein (sST2) that is thought to be a decoy receptor for IL-33 signaling. Elevated sST2 levels in serum are associated with an increased risk for cardiovascular disease. We investigated the determinants of sST2 plasma concentrations in 2,991 Framingham Offspring Cohort participants. While clinical and environmental factors explained some variation in sST2 levels, much of the variation in sST2 production was driven by genetic factors. In a genome-wide association study (GWAS), multiple SNPs within IL1RL1 (the gene encoding ST2) demonstrated associations with sST2 concentrations. Five missense variants of IL1RL1 correlated with higher sST2 levels in the GWAS and mapped to the intracellular domain of ST2, which is absent in sST2. In a cell culture model, IL1RL1 missense variants increased sST2 expression by inducing IL-33 expression and enhancing IL-33 responsiveness (via ST2L). Our data suggest that genetic variation in IL1RL1 can result in increased levels of sST2 and alter immune and inflammatory signaling through the ST2/IL-33 pathway.

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    04/12/13 | Structure, activity, and substrate selectivity of the Orf6 thioesterase from Photobacterium profundum.
    Rodríguez-Guilbe M, Oyola-Robles D, Schreiter ER, Baerga-Ortiz A
    Journal of Biological Chemistry. 2013 Apr 12;288(15):10841-8. doi: 10.1074/jbc.M112.446765

    Thioesterase activity is typically required for the release of products from polyketide synthase enzymes, but no such enzyme has been characterized in deep-sea bacteria associated with the production of polyunsaturated fatty acids. In this work, we have expressed and purified the Orf6 thioesterase from Photobacterium profundum. Enzyme assays revealed that Orf6 has a higher specific activity toward long-chain fatty acyl-CoA substrates (palmitoyl-CoA and eicosapentaenoyl-CoA) than toward short-chain or aromatic acyl-CoA substrates. We determined a high resolution (1.05 Å) structure of Orf6 that reveals a hotdog hydrolase fold arranged as a dimer of dimers. The putative active site of this structure is occupied by additional electron density not accounted for by the protein sequence, consistent with the presence of an elongated compound. A second crystal structure (1.40 Å) was obtained from a crystal that was grown in the presence of Mg(2+), which reveals the presence of a binding site for divalent cations at a crystal contact. The Mg(2+)-bound structure shows localized conformational changes (root mean square deviation of 1.63 Å), and its active site is unoccupied, suggesting a mechanism to open the active site for substrate entry or product release. These findings reveal a new thioesterase enzyme with a preference for long-chain CoA substrates in a deep-sea bacterium whose potential range of applications includes bioremediation and the production of biofuels.

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    11/01/11 | The structure, molecular dynamics, and energetics of centrin-melittin complex.
    Sosa LD, Alfaro E, Santiago J, Narváez D, Rosado MC, Rodríguez A, Gómez AM, Schreiter ER, Pastrana-Ríos B
    Proteins. 2011 Nov;79(11):3132-43. doi: 10.1002/prot.23142

    Centrin is a calcium binding protein (CaBP) belonging to the EF-hand superfamily. As with other proteins within this family, centrin is a calcium sensor with multiple biological target proteins. We chose to study Chlamydomonas reinhardtii centrin (Crcen) and its interaction with melittin (MLT) as a model for CaBP complexes due to its amphipathic properties. Our goal was to determine the molecular interactions that lead to centrin-MLT complex formation, their relative stability, and the conformational changes associated with the interaction, when compared to the single components. For this, we determined the thermodynamic parameters that define Crcen-MLT complex formation. Two-dimensional infrared (2D IR) correlation spectroscopy were used to study the amide I', I'*, and side chain bands for (13)C-Crcen, MLT, and the (13)C-Crcen-MLT complex. This approach resulted in the determination of MLT's increased helicity, while centrin was stabilized within the complex. Herein we provide the first complete molecular description of centrin-MLT complex formation and the dissociation process. Also, discussed is the first structure of a CaBP-MLT complex by X-ray crystallography, which shows that MLT has a different binding orientation than previously characterized centrin-bound peptides. Finally, all of the experimental results presented herein are consistent with centrin maintaining an extended conformation while interacting with MLT. The molecular implications of these results are: (1) the recognition of hydrophobic contacts as requirements for initial binding, (2) minimum electrostatic interactions within the C-terminal end of the peptide, and (3) van der Waals interactions within MLTs N-terminal end are required for complex formation.

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    09/14/10 | Structural basis of low-affinity nickel binding to the nickel-responsive transcription factor NikR from Escherichia coli.
    Phillips CM, Schreiter ER, Stultz CM, Drennan CL
    Biochemistry. 2010 Sep 14;49(36):7830-8. doi: 10.1021/bi100923j

    Escherichia coli NikR regulates cellular nickel uptake by binding to the nik operon in the presence of nickel and blocking transcription of genes encoding the nickel uptake transporter. NikR has two binding affinities for the nik operon: a nanomolar dissociation constant with stoichiometric nickel and a picomolar dissociation constant with excess nickel [Bloom, S. L., and Zamble, D. B. (2004) Biochemistry 43, 10029-10038; Chivers, P. T., and Sauer, R. T. (2002) Chem. Biol. 9, 1141-1148]. While it is known that the stoichiometric nickel ions bind at the NikR tetrameric interface [Schreiter, E. R., et al. (2003) Nat. Struct. Biol. 10, 794-799; Schreiter, E. R., et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 13676-13681], the binding sites for excess nickel ions have not been fully described. Here we have determined the crystal structure of NikR in the presence of excess nickel to 2.6 A resolution and have obtained nickel anomalous data (1.4845 A) in the presence of excess nickel for both NikR alone and NikR cocrystallized with a 30-nucleotide piece of double-stranded DNA containing the nik operon. These anomalous data show that excess nickel ions do not bind to a single location on NikR but instead reveal a total of 22 possible low-affinity nickel sites on the NikR tetramer. These sites, for which there are six different types, are all on the surface of NikR, and most are found in both the NikR alone and NikR-DNA structures. Using a combination of crystallographic data and molecular dynamics simulations, the nickel sites can be described as preferring octahedral geometry, utilizing one to three protein ligands (typically histidine) and at least two water molecules.

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    07/01/10 | The heparin-binding domain of HB-EGF mediates localization to sites of cell-cell contact and prevents HB-EGF proteolytic release.
    Prince RN, Schreiter ER, Zou P, Wiley HS, Ting AY, Lee RT, Lauffenburger DA
    Journal of Cell Science. 2010 Jul 1;123(Pt 13):2308-18. doi: 10.1242/jcs.058321

    Heparin-binding EGF-like growth factor (HB-EGF) is a ligand for EGF receptor (EGFR) and possesses the ability to signal in juxtacrine, autocrine and/or paracrine mode, with these alternatives being governed by the degree of proteolytic release of the ligand. Although the spatial range of diffusion of released HB-EGF is restricted by binding heparan-sulfate proteoglycans (HSPGs) in the extracellular matrix and/or cellular glycocalyx, ascertaining mechanisms governing non-released HB-EGF localization is also important for understanding its effects. We have employed a new method for independently tracking the localization of the extracellular EGF-like domain of HB-EGF and the cytoplasmic C-terminus. A striking observation was the absence of the HB-EGF transmembrane pro-form from the leading edge of COS-7 cells in a wound-closure assay; instead, this protein localized in regions of cell-cell contact. A battery of detailed experiments found that this localization derives from a trans interaction between extracellular HSPGs and the HB-EGF heparin-binding domain, and that disruption of this interaction leads to increased release of soluble ligand and a switch in cell phenotype from juxtacrine-induced growth inhibition to autocrine-induced proliferation. Our results indicate that extracellular HSPGs serve to sequester the transmembrane pro-form of HB-EGF at the point of cell-cell contact, and that this plays a role in governing the balance between juxtacrine versus autocrine and paracrine signaling.

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    09/11/09 | Thioredoxin-independent regulation of metabolism by the alpha-arrestin proteins.
    Patwari P, Chutkow WA, Cummings K, Verstraeten VL, Lammerding J, Schreiter ER, Lee RT
    Journal of Biological Chemistry. 2009 Sep 11;284(37):24996-5003. doi: 10.1074/jbc.M109.018093

    Thioredoxin-interacting protein (Txnip), originally characterized as an inhibitor of thioredoxin, is now known to be a critical regulator of glucose metabolism in vivo. Txnip is a member of the alpha-arrestin protein family; the alpha-arrestins are related to the classical beta-arrestins and visual arrestins. Txnip is the only alpha-arrestin known to bind thioredoxin, and it is not known whether the metabolic effects of Txnip are related to its ability to bind thioredoxin or related to conserved alpha-arrestin function. Here we show that wild type Txnip and Txnip C247S, a Txnip mutant that does not bind thioredoxin in vitro, both inhibit glucose uptake in mature adipocytes and in primary skin fibroblasts. Furthermore, we show that Txnip C247S does not bind thioredoxin in cells, using thiol alkylation to trap the Txnip-thioredoxin complex. Because Txnip function was independent of thioredoxin binding, we tested whether inhibition of glucose uptake was conserved in the related alpha-arrestins Arrdc4 and Arrdc3. Both Txnip and Arrdc4 inhibited glucose uptake and lactate output, while Arrdc3 had no effect. Structure-function analysis indicated that Txnip and Arrdc4 inhibit glucose uptake independent of the C-terminal WW-domain binding motifs, recently identified as important in yeast alpha-arrestins. Instead, regulation of glucose uptake was intrinsic to the arrestin domains themselves. These data demonstrate that Txnip regulates cellular metabolism independent of its binding to thioredoxin and reveal the arrestin domains as crucial structural elements in metabolic functions of alpha-arrestin proteins.

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    05/01/08 | Crystallization and preliminary X-ray characterization of full-length Chlamydomonas reinhardtii centrin.
    Alfaro E, Sosa LD, Sanoguet Z, Pastrana-Ríos B, Schreiter ER
    Acta Crystallographica Section F Structural Biology and Crystallization Communications. 2008 May 1;64(Pt 5):402-4. doi: 10.1107/S1744309108009123

    Chlamydomonas reinhardtii centrin is a member of the EF-hand calcium-binding superfamily. It is found in the basal body complex and is important for flagellar motility. Like other members of the EF-hand family, centrin interacts with and modulates the function of other proteins in a calcium-dependent manner. To understand how C. reinhardtii centrin interacts with its protein targets, it has been crystallized in the presence of the model peptide melittin and X-ray diffraction data have been collected to 2.2 A resolution. The crystals are orthorhombic, with unit-cell parameters a = 52.1, b = 114.4, c = 34.8 A, and are likely to belong to space group P2(1)2(1)2.

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    02/19/08 | Structural basis of the metal specificity for nickel regulatory protein NikR.
    Phillips CM, Schreiter ER, Guo Y, Wang SC, Zamble DB, Drennan CL
    Biochemistry. 2008 Feb 19;47(7):1938-46. doi: 10.1021/bi702006h

    In the presence of excess nickel, Escherichia coli NikR regulates cellular nickel uptake by suppressing the transcription of the nik operon, which encodes the nickel uptake transporter, NikABCDE. Previously published in vitro studies have shown that NikR is capable of binding a range of divalent transition metal ions in addition to Ni2+, including Co2+, Cu2+, Zn2+, and Cd2+. To understand how the high-affinity nickel binding site of NikR is able to accommodate these other metal ions, and to improve our understanding of NikR's mechanism of binding to DNA, we have determined structures of the metal-binding domain (MBD) of NikR in the apo form and in complex with Cu2+ and Zn2+ ions and compared them with the previously published structures with Ni2+. We observe that Cu2+ ions bind in a manner very similar to that of Ni2+, with a square planar geometry but with longer bond lengths. Crystals grown in the presence of Zn2+ reveal a protein structure similar to that of apo MBD with a disordered alpha3 helix, but with two electron density peaks near the Ni2+ binding site corresponding to two Zn2+ ions. These structural findings along with biochemical data on NikR support a hypothesis that ordering of the alpha3 helix is important for repressor activation.

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    09/01/07 | Ribbon-helix-helix transcription factors: variations on a theme.
    Schreiter ER, Drennan CL
    Nature Reviews Microbiology. 2007 Sep;5(9):710-20. doi: 10.1038/nrmicro1717

    The ribbon-helix-helix (RHH) superfamily of transcription factors uses a conserved three-dimensional structural motif to bind to DNA in a sequence-specific manner. This functionally diverse protein superfamily regulates the transcription of genes that are involved in the uptake of metals, amino-acid biosynthesis, cell division, the control of plasmid copy number, the lytic cycle of bacteriophages and, perhaps, many other cellular processes. In this Analysis, the structures of different RHH transcription factors are compared in order to evaluate the sequence motifs that are required for RHH-domain folding and DNA binding, as well as to identify conserved protein-DNA interactions in this superfamily.

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    07/06/07 | S-nitrosylation-induced conformational change in blackfin tuna myoglobin.
    Schreiter ER, Rodríguez MM, Weichsel A, Montfort WR, Bonaventura J
    Journal of Biological Chemistry. 2007 Jul 6;282(27):19773-80. doi: 10.1074/jbc.M701363200

    S-nitrosylation is a post-translational protein modification that can alter the function of a variety of proteins. Despite the growing wealth of information that this modification may have important functional consequences, little is known about the structure of the moiety or its effect on protein tertiary structure. Here we report high-resolution x-ray crystal structures of S-nitrosylated and unmodified blackfin tuna myoglobin, which demonstrate that in vitro S-nitrosylation of this protein at the surface-exposed Cys-10 directly causes a reversible conformational change by "wedging" apart a helix and loop. Furthermore, we have demonstrated in solution and in a single crystal that reduction of the S-nitrosylated myoglobin with dithionite results in NO cleavage from the sulfur of Cys-10 and rebinding to the reduced heme iron, showing the reversibility of both the modification and the conformational changes. Finally, we report the 0.95-A structure of ferrous nitrosyl myoglobin, which provides an accurate structural view of the NO coordination geometry in the context of a globin heme pocket.

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