Structural Basis for Ligand-Regulated Oligomerization of AraC

Soisson, S.M.; MacDougall-Shackleton, Beth; Schleif, Robert; Wolberger, Cynthia

doi:10.1126/science.276.5311.421

Cited by 207 publications

(306 citation statements)

References 26 publications

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“…The L-ara complexed structure revealed primary contacts between a single L-ara molecule in the ligand binding domain and residues P8, T24, R38, Y82, and H93, as well as several other residues indirectly interacting with L-ara through waterbridged hydrogen bonds. 10 In addition, substantial conformational changes in the wt-AraC N-terminal arm (residues 1-18) upon ligand binding were observed. 11,12 Substitutions at residue F15 dramatically affect the response to L-ara, resulting in constitutive and noninducible AraC variants.…”

Section: Statement Of Significancementioning

confidence: 99%

“…10,13,16 To better understand the roles of the five AraC-TAL1 amino acid substitutions and assess their potential cooperative effects, we investigated the TAL and L-ara responses of 32 AraC variants representing all combinations of wt-AraC or AraC-TAL1 residues, at the five target residues (Table III). Other than wt-AraC and AraC-TAL1, only three variants retain partial responses (>15% of wt-AraC or AraC-TAL to L-ara or TAL, respectively).…”

Section: Amino Acid Substitutions In Arac-tal Variants Reveal Mostly mentioning

confidence: 99%

“…wt-AraC also exhibits a dimer through a "b-kiss" interface but only in the apo form, where a tyrosine (Y31) of the adjacent monomer fills the ligand-binding pocket and interacts with W95. 10,19 However, the b-kiss of AraC-TAL1 is slightly rotated relative to the apo wt-AraC b-kiss, which prevents Y31 from filling the ligand pocket and interacting with W95. Overall, the structure of apo-AraC-TAL1 suggests the regulatory mechanism is similar to that of wt-AraC but with a modified binding pocket that accepts different substrates.…”

Section: Structure Of Arac-tal1 Ligand-binding Domain Reveals Differementioning

confidence: 99%

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Analysis of amino acid substitutions in AraC variants that respond to triacetic acid lactone

et al. 2016

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The Escherichia coli regulatory protein AraC regulates expression of ara genes in response to L-arabinose. In efforts to develop genetically encoded molecular reporters, we previously engineered an AraC variant that responds to the compound triacetic acid lactone (TAL). This variant (named "AraC-TAL1") was isolated by screening a library of AraC variants, in which five amino acid positions in the ligand-binding pocket were simultaneously randomized. Screening was carried out through multiple rounds of alternating positive and negative fluorescence-activated cell sorting. Here we show that changing the screening protocol results in the identification of different TAL-responsive variants (nine new variants). Individual substituted residues within these variants were found to primarily act cooperatively toward the gene expression response. Finally, X-ray diffraction was used to solve the crystal structure of the apo AraC-TAL1 ligand-binding domain. The resolved crystal structure confirms that this variant takes on a structure nearly identical to the apo wild-type AraC ligand-binding domain (root-mean-square deviation 0.93 Å ), suggesting that AraC-TAL1 behaves similar to wild-type with regard to ligand recognition and gene regulation. Our results provide amino acid sequence-function data sets for training and validating AraC modeling studies, and contribute to our understanding of how to design new biosensors based on AraC.

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Section: Statement Of Significancementioning

confidence: 99%

Section: Amino Acid Substitutions In Arac-tal Variants Reveal Mostly mentioning

confidence: 99%

Section: Structure Of Arac-tal1 Ligand-binding Domain Reveals Differementioning

confidence: 99%

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Analysis of amino acid substitutions in AraC variants that respond to triacetic acid lactone

et al. 2016

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“…Notably, the C-terminal domain of Pirin does not contain a metal binding site and its sequence does not contain the conserved metal-coordinating residues. Several members of the cupin superfamily do not contain the metal-coordinating residues, including the transcription factor araC from Escherichia coli (21). araC is a single domain member of the cupin family and binds arabinose for activation of transcription.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of Human Pirin

Pang

Bartlam

Zeng

et al. 2004

Journal of Biological Chemistry

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Pirin is a newly identified nuclear protein that interacts with the oncoprotein B-cell lymphoma 3-encoded (Bcl-3) and nuclear factor I (NFI). The crystal structure of human Pirin at 2.1-Å resolution shows it to be a member of the functionally diverse cupin superfamily. The structure comprises two ␤-barrel domains, with an Fe(II) cofactor bound within the cavity of the N-terminal domain. These findings suggest an enzymatic role for Pirin, most likely in biological redox reactions involving oxygen, and provide compelling evidence that Pirin requires the participation of the metal ion for its interaction with Bcl-3 to co-regulate the NF-B transcription pathway and the interaction with NFI in DNA replication. Substitution of iron by heavy metals thus provides a novel pathway for these metals to directly influence gene transcription. The structure suggests an interesting new role of iron in biology and that Pirin may be involved in novel mechanisms of gene regulation.Pirin is a newly identified nuclear protein that is widely expressed in dot-like subnuclear structures in human tissues, in particular liver and heart (1). Pirins are highly conserved among mammals, plants, fungi, and even prokaryotic organisms and have been assigned as a sub-family of the cupin superfamily based on both structure and sequence homology (1, 2). The cupin superfamily is among the most functionally diverse of any protein family described to date, with both enzymatic and non-enzymatic members included (2). This cupin superfamily is grouped according to a conserved ␤-barrel fold and two characteristic sequence motifs. Study of Pirin reveals two cupin domains from its primary sequence that are consistent with other members of the superfamily.The exact functions of Pirins are not yet known. No enzymatic activity has been described, but human Pirin has been found to bind to the nuclear factor I/CCAAT box transcription factor (NFI) 1 and to the oncoprotein B cell lymphoma 3-encoded (Bcl-3) in vivo (1, 3), suggesting that it is a transcription cofactor. NFI is known to stimulate DNA replication and RNA polymerase II-driven transcription (4). Bcl-3 is a distinctive member of the I B family, which inhibits the transcription factor NF-B by preventing NF-B nuclear translocation and DNA binding. However, there is also evidence that Bcl-3 preferentially binds to NF-B p50 or p52 homodimers to stimulate transcription (5). The functional nature of this difference between I B (inhibiting) and Bcl-3 (enhancing) is not known, but it is clear that they bind to different protein partners. Pirin is one of four binding partners of Bcl-3, together with Bard1, Tip60, and Jab1, and can be sequestered into quaternary complexes with Bcl-3, p50, and DNA (3). These four Bcl-3-interacting protein partners, which do not share any sequence homology, might play some crucial role in regulating the function of Bcl-3 and I B. Indeed, both Pirin and Bcl-3 are localized in the nucleus, and the potential roles of Pirin in NF-B-dependent transcriptional regulation are implicated i...

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“…This is the case for AraC and arabinose (82) or UreR and urea (83) for examples. In addition to the interaction with small molecules, the non-conserved domain may establish productive contacts with the RNA polymerase holoenzyme and increase the affinity constant or the isomerization of the close complex into an open complex.…”

Section: Discussionmentioning

confidence: 99%

Mycobacterium tuberculosis Rv1395 Is a Class III Transcriptional Regulator of the AraC Family Involved in Cytochrome P450 Regulation

Recchi

Sclavi

Rauzier

et al. 2003

Journal of Biological Chemistry

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Rv1395 is annotated as a potential transcriptional regulator of the AraC family. The Rv1395 insertional mutant was identified in a signature tag mutagenesis study in Mycobacterium tuberculosis and was shown to be attenuated in the lungs of mice. Here, we used comparative genomics and biochemical methods to show that Rv1395 is unique to the M. tuberculosis complex and that it encodes a protein that binds the region between two divergent genes, a member of the cytochrome P450 family (Rv1394c or cyp132) and Rv1395 itself. Rv1395 binds to this DNA region by its helix-turn-helix-containing Cterminal domain, and it recognizes two sites with different affinity. We identified the transcriptional start points (TSP) of Rv1394c and Rv1395: both genes have two TSPs, three of which are located in the intergenic region. We constructed and compared various transcriptional fusions consisting of the promoter regions and a reporter gene in Mycobacterium smegmatis: this showed that Rv1395 induces the expression of the cytochrome P450 gene (Rv1394c) and represses its own transcription. This was confirmed in M. tuberculosis when the wild type and a Rv1395-overexpressing strain were used as hosts for the fusions. Site-directed mutagenesis showed that Rv1395 binds to the two sites in a co-operative manner and that binding to both sites is required for Rv1395 optimal activity. A model describing the potential mode of action of Rv1395 is discussed.

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Structural Basis for Ligand-Regulated Oligomerization of AraC

Cited by 207 publications

References 26 publications

Analysis of amino acid substitutions in AraC variants that respond to triacetic acid lactone

Analysis of amino acid substitutions in AraC variants that respond to triacetic acid lactone

Crystal Structure of Human Pirin

Mycobacterium tuberculosis Rv1395 Is a Class III Transcriptional Regulator of the AraC Family Involved in Cytochrome P450 Regulation

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