Metal binding properties and secondary structure of the zinc-binding domain of Nup475

Worthington, Mark T.; Amann, Barbara; Nathans, Daniel; Berg, Jeremy M.

doi:10.1073/pnas.93.24.13754

Cited by 99 publications

(131 citation statements)

References 36 publications

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“…Such studies have been difficult in the past because of problems with solubility of the recombinant protein and its constituent peptides. Recent studies using a peptide from the mouse TTP tandem zinc finger domain, in which a tyrosine-tolysine mutation had been created at position 143 of the mouse protein (GenBank accession number NP_035886), suggested that whereas a structure could be determined for the first zinc finger by NMR methods, a structure could not be solved for the second zinc finger (26,27). Similarly, in the present studies, the amino acids composing the first zinc finger of the purified recombinant human TTP (102-174) peptide could be assigned, but those of the second zinc finger could not be identified, perhaps because of similar problems to those observed by Berg et al (27) with the mutant mouse peptide.…”

Section: Discussionmentioning

confidence: 99%

Characteristics of the Interaction of a Synthetic Human Tristetraprolin Tandem Zinc Finger Peptide with AU-rich Element-containing RNA Substrates

Blackshear

Lai²,

Kennington³

et al. 2003

Journal of Biological Chemistry

116

130

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Tristetraprolin (TTP) and its two known mammalian family members are tandem CCCH zinc finger proteins that can bind to AU-rich elements (AREs) in cellular mRNAs and destabilize those transcripts, apparently by initiating their deadenylation. Previous studies have shown that the ϳ70-amino acid tandem zinc finger domain of TTP is required and sufficient for RNA binding, and that the integrity of both zinc fingers is also required. However, little is known about the kinetics or structure of the peptide-RNA interaction, in part because of difficulties in obtaining soluble recombinant protein or peptides. We characterized the binding of a synthetic 73-amino acid peptide from human TTP to the tumor necrosis factor (TNF) ARE by gel mobility shift analyses and fluorescence anisotropy experiments. Both types of studies yielded a peptide-RNA dissociation constant of ϳ10 nM. Surprisingly, we found that the "footprint" from the TNF ARE required for peptide binding was only ϳ9 bases and that two molecules of peptide could bind to probes containing as little as 19 bases. An identical recombinant peptide exhibited gel shift characteristics similar to those of the synthetic peptide. NMR analysis of the 15 N-labeled recombinant peptide suggested that its first zinc finger was structured in solution but that the second was not. The titration of oligonucleotides representing 17, 13, and even 9 bases of the TNF ARE caused an essentially identical, dramatic shift of existing resonances, and the appearance of new resonances in the peptide spectra, so that all amino acids could be assigned. These data suggest that this TTP peptide-RNA complex is structured in solution and might be amenable to NMR structure determination. Tristetraprolin (TTP)1 is the prototype of a small family of eukaryotic CCCH tandem zinc finger proteins (see Ref. 1 for recent review). In mammals, there are three known members of this protein family, whereas fish and frogs express a fourth member that seems to consist of a group of closely related proteins (2-4). The characteristic tandem zinc finger domain of these proteins consists of two zinc fingers with 18 amino acids between fingers, strict intrafinger spacing of Cx8Cx5Cx3H, and a characteristic sequence motif leading into each finger of R(K)YKTEL or a closely related variant.Studies in TTP knockout mice and cells derived from them revealed that TTP acts in normal physiology to regulate the synthesis and secretion of two clinically important cytokines, tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (5-9). The increased secretion of these cytokines in the TTP knockout mice was secondary to increased steady state levels of their mRNAs, which in turn was caused by excessive stabilization of the transcripts. TTP was found to confer instability on these two transcripts by binding to the so-called class II AREs contained within the 3Ј-untranslated regions of these mRNAs (7, 10 -13). The tandem zinc finger domain was found to be the RNA binding domain, and the invariant cys...

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Section: Discussionmentioning

confidence: 99%

Characteristics of the Interaction of a Synthetic Human Tristetraprolin Tandem Zinc Finger Peptide with AU-rich Element-containing RNA Substrates

Blackshear

Lai²,

Kennington³

et al. 2003

Journal of Biological Chemistry

116

130

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“…TTP fusion protein and its tandem zinc finger peptide each bind zinc with an affinity similar to those of other wellcharacterized zinc finger peptides (17)(18)(19). Zinc binding is required for the TNF mRNA ARE binding activity of TTP (9).…”

mentioning

confidence: 99%

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications^,

Cao

2004

Biochemistry

122

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Tristetraprolin (TTP) is a hyperphosphorylated protein that destabilizes mRNA by binding to an AUrich element (ARE). Mice deficient in TTP develop a severe inflammatory syndrome. The biochemical properties of TTP have not been adequately characterized, due to the difficulties in protein purification and lack of a high-titer antiserum. Full-length human TTP was expressed in human HEK293 cells and purified to at least 70% homogeneity. The purified protein was free of endogenous ARE binding activity, and was used for investigating its size, zinc dependency, and binding kinetics for tumor necrosis factor α mRNA ARE. A high-titer rabbit antiserum was raised against the MBP-hTTP fusion protein expressed in Escherichia coli. Cellular localization studies of the transfected cells indicated that approximately 80% of the expressed TTP was in the cytosol, with 20% in the nuclei. TTP from both locations bound to the ARE and formed similar complexes. The purified TTP was shown to be intact by N-terminal His-tag purification, C-terminal peptide sequencing, and mass spectrometry analysis. Results from size exclusion chromatography are consistent with the predominant form of active TTP being a tetramer. TTP's ARE binding activity was increased by 10 μM Zn 2+ . The half-maximal binding of TTP from HEK293 cells was approximately 30 nM in assays containing 10 nM ARE. This value was about twice that of TTP from E. coli. TTP from HEK293 cells was highly phosphorylated, and its electrophoretic mobility was increased by alkaline phosphatase treatment and somewhat by T271A mutation, but not by PNGase F or S186A mutation. The gel mobility of TTP from E. coli was decreased by in vitro phosphorylation with p42/ERK2 and p38 mitogen-activated protein kinases. These results suggest that TTP's zincdependent ARE binding affinity is reduced by half by posttranslational modifications, mainly by phosphorylation but not by glycosylation, in mammalian cells. The results support a model in which each subunit of the TTP tetramer binds to one of the five overlapping UUAUUUAUU sequences of the ARE, resulting in a stable TTP-ARE complex. Tristetraprolin (TTP)1 (also known as ZFP36, Nup475, TIS11, and G0S24) is a prototypical member of a small family of mammalian proteins with tandem CCCH (CX 8 CX 5 CX 3 H) zinc finger motifs separated by 18 amino acid residues (1). Similar zinc finger sequences are found in at least 19 species in the GenBank database, including human, cow, mouse, rat, sheep,

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“…Metal binding by His residues involves donating imidazole nitrogen lone-pair electrons to the unfilled orbitals of the metal, and cysteines can substitute for His in this process. 38,[41][42][43] Thus, mutants His267Cys, His272Cys, and His267Cys/His272Cys were created. The imidazole proton of His has a pK a of 6.6 and can mediate pH-dependent effects in proteins.…”

Section: Conserved Histidine Pair In Tm6mentioning

confidence: 99%

Iron transport by Nramp2/DMT1: pH regulation of transport by 2 histidines in transmembrane domain 6

Lam-Yuk-Tseung

Govoni

Forbes

et al. 2003

Blood

103

102

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Mutations at natural resistance-associated macrophage protein 1 (Nramp1) impair phagocyte function and cause susceptibility to infections while mutations at Nramp2 (divalent metal transporter 1 [DMT1]) affect iron homeostasis and cause severe microcytic anemia. Structure-function relationships in the Nramp superfamily were studied by mutagenesis, followed by functional characterization in yeast and in mammalian cells. These studies identify 3 negatively charged and highly conserved residues in transmembrane domains (TM) 1, 4, and 7 as essential for cation transport by Nramp2/DMT1. The introduction of a charged residue (Gly185Arg) in TM4 found in the naturally occurring microcytic anemia mk (mouse) and Belgrade (rat) mutants is shown to cause a partial or complete loss of function in mammalian and yeast cells, respectively. A pair of mutation-sensitive and highly conserved histidines (His267, His272) was identified in TM6. Surprisingly, inactive His267 and His272 mutants could be rescued by lowering the pH of the transport assay. This indicates that His267/His272 are not directly involved in metal binding but, rather, play an important role in pH regulation of metal transport by Nramp proteins.

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Metal binding properties and secondary structure of the zinc-binding domain of Nup475

Cited by 99 publications

References 36 publications

Characteristics of the Interaction of a Synthetic Human Tristetraprolin Tandem Zinc Finger Peptide with AU-rich Element-containing RNA Substrates

Characteristics of the Interaction of a Synthetic Human Tristetraprolin Tandem Zinc Finger Peptide with AU-rich Element-containing RNA Substrates

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications^,

Iron transport by Nramp2/DMT1: pH regulation of transport by 2 histidines in transmembrane domain 6

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Metal binding properties and secondary structure of the zinc-binding domain of Nup475

Cited by 99 publications

References 36 publications

Characteristics of the Interaction of a Synthetic Human Tristetraprolin Tandem Zinc Finger Peptide with AU-rich Element-containing RNA Substrates

Characteristics of the Interaction of a Synthetic Human Tristetraprolin Tandem Zinc Finger Peptide with AU-rich Element-containing RNA Substrates

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications,

Iron transport by Nramp2/DMT1: pH regulation of transport by 2 histidines in transmembrane domain 6

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Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications^,