The ChiS family DNA-binding domain contains a cryptic helix-turn-helix variant

AbstractSequence specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen Vibrio cholerae, the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We have recently shown that ChiS contains a cryptic DBD in its C-terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 Å. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight “turn” of the HTH is replaced with an extended loop containing a β-sheet, a variant which we term the “helix-sheet-helix”. Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse Proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs.ImportanceRegulating gene expression is essential in all domains of life. This process is commonly facilitated by the activity of DNA-binding transcription factors. There are diverse structural domains that allow proteins to bind to specific DNA sequences. The structural basis underlying how some proteins bind to DNA, however, remains unclear. Previously, we showed that in the major human pathogen Vibrio cholerae, the transcription factor ChiS directly regulates gene expression through a cryptic DNA binding domain. This domain lacked homology to any known DNA-binding protein. In the current study, we determined the structure of the ChiS DNA binding domain (DBD) and find that the ChiS-family DBD is a cryptic variant of the ubiquitous helix-turn-helix (HTH) domain. We further demonstrate that this domain is conserved in diverse proteins that may represent a novel group of transcriptional regulators.

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The ChiS-Family DNA-Binding Domain Contains a Cryptic Helix-Turn-Helix Variant

mBio ◽

10.1128/mbio.03287-20 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Catherine A. Klancher ◽

George Minasov ◽

Ram Podicheti ◽

Douglas B. Rusch ◽

Triana N. Dalia ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Vibrio Cholerae ◽

Binding Domain ◽

Structural Basis ◽

Striking Difference ◽

Dna Binding Domain ◽

Structural Domains ◽

Content Type ◽

Domains Of Life

ABSTRACT Sequence-specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen Vibrio cholerae, the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We recently showed that ChiS contains a cryptic DBD in its C terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 Å. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA-binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight turn of the HTH is replaced with an insertion containing a β-sheet, a variant which we term the helix-sheet-helix. Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs. IMPORTANCE Regulating gene expression is essential in all domains of life. This process is commonly facilitated by the activity of DNA-binding transcription factors. There are diverse structural domains that allow proteins to bind to specific DNA sequences. The structural basis underlying how some proteins bind to DNA, however, remains unclear. Previously, we showed that in the major human pathogen Vibrio cholerae, the transcription factor ChiS directly regulates gene expression through a cryptic DNA-binding domain. This domain lacked homology to any known DNA-binding protein. In the current study, we determined the structure of the ChiS DNA-binding domain (DBD) and found that the ChiS-family DBD is a cryptic variant of the ubiquitous helix-turn-helix (HTH) domain. We further demonstrate that this domain is conserved in diverse proteins that may represent a novel group of transcriptional regulators.

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Mechanism underlying the DNA-binding preferences of the Vibrio cholerae and vibriophage VP882 VqmA quorum-sensing receptors

10.1101/2021.04.15.439952 ◽

2021 ◽

Author(s):

Bonnie L. Bassler ◽

Olivia Duddy ◽

Xiuliang Huang ◽

Justin Silpe

Keyword(s):

Dna Binding ◽

Quorum Sensing ◽

Vibrio Cholerae ◽

Dna Sequences ◽

Genetic Screen ◽

Binding Domain ◽

Communication Process ◽

Dna Binding Domain ◽

Phage Gene ◽

Small Regulatory Rna

Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. In the global pathogen Vibrio cholerae, one quorum-sensing receptor and transcription factor, called VqmA (VqmAVc), activates expression of the vqmR gene encoding the small regulatory RNA VqmR, which represses genes involved in virulence and biofilm formation. Vibriophage VP882 encodes a VqmA homolog called VqmAPhage that activates transcription of the phage gene qtip, and Qtip launches the phage lytic program. Curiously, VqmAPhage can activate vqmR expression but VqmAVc cannot activate expression of qtip. Here, we investigate the mechanism underlying this asymmetry. We find that promoter selectivity is driven exclusively by each VqmA DNA-binding domain and key DNA sequences in the vqmR and qtip promoters are required to maintain specificity. A protein sequence-guided mutagenesis approach revealed that the residue E194 of VqmAPhage and A192, the equivalent residue in VqmAVc, in the helix-turn-helix motifs contribute to promoter-binding specificity. A genetic screen to identify VqmAPhage mutants that are incapable of binding the qtip promoter but maintain binding to the vqmR promoter delivered additional VqmAPhage residues located immediately C-terminal to the helix-turn-helix motif as required for binding the qtip promoter. Surprisingly, these residues are conserved between VqmAPhage and VqmAVc. A second, targeted genetic screen revealed a region located in the VqmAVc DNA-binding domain as necessary to prevent VqmAVc from binding the qtip promoter, thus restricting DNA-binding to the vqmR promoter. We propose that the VqmAVc helix-turn-helix motif and the C-terminal flanking residues function together to prohibit VqmAVc from binding the qtip promoter.

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The regulatory function of dIno80 correlates with its DNA binding activity

10.1101/519959 ◽

2019 ◽

Cited By ~ 1

Author(s):

S Jain ◽

J Maini ◽

A Narang ◽

S Maiti ◽

V Brahmachari

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Dna Sequences ◽

Specific Binding ◽

Binding Domain ◽

Regulatory Function ◽

Binding Potential ◽

Homeotic Genes ◽

Dna Binding Domain

ABSTRACTThe INO80 complex, including the Ino80 protein, forms a highly conserved canonical complex that remodels chromatin in the context of multiple cellular functions. TheDrosophilahomologue, dIno80, is involved in homeotic gene regulation during development as a canonical Pho-dIno80 complex. Previously, we found that dIno80 regulates homeotic genes by interacting with epigenetic regulators, such as polycomb and trithorax, suggesting the occurrence of non-canonical Ino80 complexes. Here using spectroscopic methods and gel retardation assays, we identified a set of consensus DNA sequences that DNA binding domain of dIno80 (DBINO) interacts with having differential affinity and high specificity. Testing these sequences in reporter assays, showed that this interaction can positively regulate transcription. These results suggest that, dIno80 has a sequence preference for interaction with DNA leading to transcriptional changes.SIGNIFICANCEThe chromatin remodeling proteins control gene expression by nucleosome sliding and exchange. They are known to function as multi-subunit complexes recruited to chromatin by transcription factors or histone modification readers. Here, we report a sequence specific binding potential for the chromatin remodeler, dIno80. We have carried outin vitrostudies with DNA binding domain of dIno80 to elucidate its sequence specific DNA binding. We have also showed that this binding can regulated reporter gene expression inDrosophilacells. Our results suggest a non-canonical role of Ino80 in transcriptional regulation.

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Mechanism underlying the DNA-binding preferences of the Vibrio cholerae and vibriophage VP882 VqmA quorum-sensing receptors

PLoS Genetics ◽

10.1371/journal.pgen.1009550 ◽

2021 ◽

Vol 17 (7) ◽

pp. e1009550

Author(s):

Olivia P. Duddy ◽

Xiuliang Huang ◽

Justin E. Silpe ◽

Bonnie L. Bassler

Keyword(s):

Dna Binding ◽

Quorum Sensing ◽

Vibrio Cholerae ◽

Dna Sequences ◽

Genetic Screen ◽

Binding Domain ◽

Communication Process ◽

Dna Binding Domain ◽

Phage Gene ◽

Small Regulatory Rna

Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. In the global pathogen Vibrio cholerae, one quorum-sensing receptor and transcription factor, called VqmA (VqmAVc), activates expression of the vqmR gene encoding the small regulatory RNA VqmR, which represses genes involved in virulence and biofilm formation. Vibriophage VP882 encodes a VqmA homolog called VqmAPhage that activates transcription of the phage gene qtip, and Qtip launches the phage lytic program. Curiously, VqmAPhage can activate vqmR expression but VqmAVc cannot activate expression of qtip. Here, we investigate the mechanism underlying this asymmetry. We find that promoter selectivity is driven by each VqmA DNA-binding domain and key DNA sequences in the vqmR and qtip promoters are required to maintain specificity. A protein sequence-guided mutagenesis approach revealed that the residue E194 of VqmAPhage and A192, the equivalent residue in VqmAVc, in the helix-turn-helix motifs contribute to promoter-binding specificity. A genetic screen to identify VqmAPhage mutants that are incapable of binding the qtip promoter but maintain binding to the vqmR promoter delivered additional VqmAPhage residues located immediately C-terminal to the helix-turn-helix motif as required for binding the qtip promoter. Surprisingly, these residues are conserved between VqmAPhage and VqmAVc. A second, targeted genetic screen revealed a region located in the VqmAVc DNA-binding domain that is necessary to prevent VqmAVc from binding the qtip promoter, thus restricting DNA-binding to the vqmR promoter. We propose that the VqmAVc helix-turn-helix motif and the C-terminal flanking residues function together to prohibit VqmAVc from binding the qtip promoter.

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Structural basis of the p53 DNA binding domain and PUMA complex

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2021.02.049 ◽

2021 ◽

Vol 548 ◽

pp. 39-46

Author(s):

Chang Woo Han ◽

Han Na Lee ◽

Mi Suk Jeong ◽

So Young Park ◽

Se Bok Jang

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Structural Basis ◽

Dna Binding Domain

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Steroid receptor-mediated inhibition of rat prolactin gene expression does not require the receptor DNA-binding domain

Cell ◽

10.1016/0092-8674(88)90406-0 ◽

1988 ◽

Vol 52 (5) ◽

pp. 685-695 ◽

Cited By ~ 128

Author(s):

Stuart Adler ◽

Marian L. Waterman ◽

Xi He ◽

Michael G. Rosenfeld

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Steroid Receptor ◽

Binding Domain ◽

Dna Binding Domain ◽

Prolactin Gene

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Adaptive evolution of transcription factor binding affinities

10.1101/118455 ◽

2017 ◽

Author(s):

Jungeui Hong ◽

Nathan Brandt ◽

Ally Yang ◽

Tim Hughes ◽

David Gresham

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Dna Binding ◽

Adaptive Evolution ◽

Consensus Sequence ◽

Binding Domain ◽

Dna Binding Domain ◽

Missense Mutations ◽

Binding Affinities ◽

Synonymous Mutations

Understanding the molecular basis of gene expression evolution is a central problem in evolutionary biology. However, connecting changes in gene expression to increased fitness, and identifying the functional basis of those changes, remains challenging. To study adaptive evolution of gene expression in real time, we performed long term experimental evolution (LTEE) of Saccharomyces cerevisiae (budding yeast) in ammonium-limited chemostats. Following several hundred generations of continuous selection we found significant divergence of nitrogen-responsive gene expression in lineages with increased fitness. In multiple independent lineages we found repeated selection for non-synonymous mutations in the zinc finger DNA binding domain of the activating transcription factor (TF), GAT1, that operates within incoherent feedforward loops to control expression of the nitrogen catabolite repression (NCR) regulon. Missense mutations in the DNA binding domain of GAT1 reduce its binding affinity for the GATAA consensus sequence in a promoter-specific manner, resulting in increased expression of ammonium permease genes via both direct and indirect effects, thereby conferring increased fitness. We find that altered transcriptional output of the NCR regulon results in antagonistic pleiotropy in alternate environments and that the DNA binding domain of GAT1 is subject to purifying selection in natural populations. Our study shows that adaptive evolution of gene expression can entail tuning expression output by quantitative changes in TF binding affinities while maintaining the overall topology of a gene regulatory network.

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AguR Is Required for Induction of the Streptococcus mutans Agmatine Deiminase System by Low pH and Agmatine

Applied and Environmental Microbiology ◽

10.1128/aem.02145-08 ◽

2009 ◽

Vol 75 (9) ◽

pp. 2629-2637 ◽

Cited By ~ 32

Author(s):

Yaling Liu ◽

Lin Zeng ◽

Robert A. Burne

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Streptococcus Mutans ◽

Low Ph ◽

Binding Domain ◽

Negative Regulator ◽

Dna Binding Domain ◽

Mutant Proteins ◽

Acidic Conditions ◽

Agmatine Deiminase

ABSTRACT Acidic conditions and the presence of exogenous agmatine are required to achieve maximal expression of the agmatine deiminase system (AgDS) of Streptococcus mutans. Here we demonstrate that the transcriptional activator of the AgDS, AguR, is required for the responses to agmatine and to low pH. Linker scanning mutagenesis was used to create a panel of mutated aguR genes that were utilized to complement an aguR deletion mutant of S. mutans. The level of production of the mutant proteins was shown to be comparable to that of the wild-type AguR protein. Mutations in the predicted DNA binding domain of AguR eliminated activation of the agu operon. Insertions into the region connecting the DNA binding domain to the predicted extracellular and transmembrane domains were well tolerated. In contrast, a variety of mutants were isolated that had a diminished capacity to respond to low pH but retained the ability to activate AgDS gene expression in response to agmatine, and vice versa. Also, a number of mutants were unable to respond to either agmatine or low pH. AguD, which is a predicted agmatine-putrescine antiporter, was found to be a negative regulator of AgDS gene expression in the absence of exogenous agmatine but was not required for low-pH induction of the AgDS genes. This study reveals that the control of AgDS gene expression by both agmatine and low pH is coordinated through the AguR protein and begins to identify domains of the protein involved in sensing and signaling.

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