Structural basis of the p53 DNA binding domain and PUMA complex

Abstract Retinoic acid receptors (RARs) as a functional heterodimer with retinoid X receptors (RXRs), bind a diverse series of RA-response elements (RAREs) in regulated genes. Among them, the non-canonical DR0 elements are bound by RXR–RAR with comparable affinities to DR5 elements but DR0 elements do not act transcriptionally as independent RAREs. In this work, we present structural insights for the recognition of DR5 and DR0 elements by RXR–RAR heterodimer using x-ray crystallography, small angle x-ray scattering, and hydrogen/deuterium exchange coupled to mass spectrometry. We solved the crystal structures of RXR–RAR DNA-binding domain in complex with the Rarb2 DR5 and RXR–RXR DNA-binding domain in complex with Hoxb13 DR0. While cooperative binding was observed on DR5, the two molecules bound non-cooperatively on DR0 on opposite sides of the DNA. In addition, our data unveil the structural organization and dynamics of the multi-domain RXR–RAR DNA complexes providing evidence for DNA-dependent allosteric communication between domains. Differential binding modes between DR0 and DR5 were observed leading to differences in conformation and structural dynamics of the multi-domain RXR–RAR DNA complexes. These results reveal that the topological organization of the RAR binding element confer regulatory information by modulating the overall topology and structural dynamics of the RXR–RAR heterodimers.

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The structural basis for recognition of base J containing DNA by a novel DNA binding domain in JBP1

Nucleic Acids Research ◽

10.1093/nar/gkr125 ◽

2011 ◽

Vol 39 (13) ◽

pp. 5715-5728 ◽

Cited By ~ 20

Author(s):

Tatjana Heidebrecht ◽

Evangelos Christodoulou ◽

Michael J. Chalmers ◽

Sabrina Jan ◽

Bas ter Riet ◽

...

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Structural Basis ◽

Dna Binding Domain

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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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Mutations in the linker domain affect phospho-STAT3 function and suggest targets for interrupting STAT3 activity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1515876112 ◽

2015 ◽

Vol 112 (48) ◽

pp. 14811-14816 ◽

Cited By ~ 18

Author(s):

Claudia Mertens ◽

Bhagwattie Haripal ◽

Sebastian Klinge ◽

James E. Darnell

Keyword(s):

Dna Binding ◽

Tyrosine Phosphorylation ◽

Transcriptional Activation ◽

Sh2 Domain ◽

Binding Domain ◽

Structural Basis ◽

Dna Binding Domain ◽

Functional Interaction ◽

Drug Candidates ◽

Linker Domain

Crystallography of the cores of phosphotyrosine-activated dimers of STAT1 (132–713) and STAT3 (127–722) bound to a similar double-stranded deoxyoligonucleotide established the domain structure of the STATs and the structural basis for activation through tyrosine phosphorylation and dimerization. We reported earlier that mutants in the linker domain of STAT1 that connect the DNA-binding domain and SH2 domain can prevent transcriptional activation. Because of the pervasive importance of persistently activated STAT3 in many human cancers and the difficulty of finding useful drug candidates aimed at disrupting the pY interchange in active STAT3 dimers, we have examined effects of an array of mutants in the STAT3 linker domain. We have found several STAT3 linker domain mutants to have profound effects of inhibiting STAT3 transcriptional activation. From these results, we propose (i) there is definite functional interaction of the linker both with the DNA binding domain and with the SH2 domain, and (ii) these putative contacts provide potential new targets for small molecule-induced pSTAT3 inhibition.

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Purification, crystallization and X-ray diffraction analysis of the DNA-binding domain of human heat-shock factor 2

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x16003599 ◽

2016 ◽

Vol 72 (4) ◽

pp. 294-299 ◽

Cited By ~ 3

Author(s):

Han Feng ◽

Wei Liu ◽

Da-Cheng Wang

Keyword(s):

Heat Shock ◽

Dna Binding ◽

Heat Shock Response ◽

Protein Family ◽

Binding Domain ◽

Structural Basis ◽

Dna Binding Domain ◽

X Ray Diffraction ◽

X Ray ◽

Shock Response

Cells respond to various proteotoxic stimuli and maintain protein homeostasis through a conserved mechanism called the heat-shock response, which is characterized by the enhanced synthesis of heat-shock proteins. This response is mediated by heat-shock factors (HSFs). Four genes encoding HSF1–HSF4 exist in the genome of mammals. In this protein family, HSF1 is the orthologue of the single HSF in lower eukaryotic organisms and is the major regulator of the heat-shock response, while HSF2, which shows low sequence homology to HSF1, serves as a developmental regulator. Increasing evidence has revealed biochemical properties and functional roles that are unique to HSF2, such as its DNA-binding preference and sumoylation patterns, which are distinct from those of HSF1. The structural basis for such differences, however, is poorly understood owing to the lack of available mammalian HSF structures. The N-terminal DNA-binding domain (DBD) is the most conserved functional module and is the only crystallizable domain in HSFs. To date, only HSF1 homologue structures from yeast and fruit fly have been determined. Along with extensive studies of the HSF family, more structural information, particularly from members with a remoter phylogenic relationship to the reported structures,e.g.HSF2, is needed in order to better understand the detailed mechanisms of HSF biology. In this work, the recombinant DBD (residues 7–112) from human HSF2 was produced inEscherichia coliand crystallized. An X-ray diffraction data set was collected to 1.32 Å resolution from a crystal belonging to space groupP212121with unit cell-parametersa= 65.66,b= 67.26,c= 93.25 Å. The data-evaluation statistics revealed good quality of the collected data, thus establishing a solid basis for the determination of the first structure at atomic resolution in this protein family.

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Expression and purification of 6xHis-tagged DNA binding domains of functional ecdysteroid receptor from drosophila melanogaster.

Acta Biochimica Polonica ◽

10.18388/abp.1996_4457 ◽

1996 ◽

Vol 43 (4) ◽

pp. 611-621 ◽

Cited By ~ 4

Author(s):

A Rusin ◽

A Niedziela-Majka ◽

G Rymarczyk ◽

A Ozyhar

Keyword(s):

Dna Binding ◽

Dna Interaction ◽

Binding Domain ◽

Structural Basis ◽

Dna Binding Domain ◽

Affinity Tag ◽

Mobility Shift ◽

Ecdysteroid Receptor ◽

Dna Binding Domains ◽

Binding Domains

Two members of the nuclear receptor superfamily, EcR and Ultraspiracle (Usp) heterodimerize to form a functional receptor for 20-hydroxyecdysone-the key ecdysteroid controlling induction and modulation of morphogenetic events through Drosophila development. In order to study aspects of receptor function and ultimately the structural basis of the ecdysteroid receptor-DNA interaction, it is necessary to produce large quantities of purified EcR and Usp DNA-binding domains. Toward this end, we have expressed the EcR DNA-binding domain and the Usp DNA-binding domain as proteins with an affinity tag consisting of six histidine residues (6xHis-EcRDBD and 6xHis-UspDBD, respectively) using the expression vector pQE-30. Under optimal conditions, elaborated in this study, bacteria can express the recombinant 6xHis-EcRDBD to the levels of 11% of total soluble proteins and the 6xHis-UspDBD to the levels of 16%. Both proteins were purified to homogeneity from the soluble protein fraction using combination of ammonium sulphate fractionation and affinity chromatography on Ni-NTA agarose. The gel mobility shift experiments demonstrated that the purified 6xHis-EcRDBD and the 6xHis-UspDBD interact specifically with an 20-hydroxyecdysone response element from the promoter region of the hsp 27 Drosophila gene.

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

10.1101/2020.11.18.389122 ◽

2020 ◽

Author(s):

Catherine A. Klancher ◽

George Minasov ◽

Ram Podicheti ◽

Douglas B. Rusch ◽

Triana N. Dalia ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Vibrio Cholerae ◽

Dna Sequences ◽

Binding Domain ◽

Structural Basis ◽

Striking Difference ◽

Dna Binding Domain ◽

Structural Domains ◽

Domains Of Life

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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Structural basis of G-quadruplex DNA recognition by the yeast telomeric protein Rap1

Nucleic Acids Research ◽

10.1093/nar/gkaa171 ◽

2020 ◽

Vol 48 (8) ◽

pp. 4562-4571 ◽

Cited By ~ 4

Author(s):

Anna Traczyk ◽

Chong Wai Liew ◽

David James Gill ◽

Daniela Rhodes

Keyword(s):

Dna Binding ◽

Dna Recognition ◽

Binding Domain ◽

Telomere Maintenance ◽

Structural Basis ◽

Dna Binding Domain ◽

Double Stranded Dna ◽

G Quadruplex ◽

Quadruplex Formation ◽

Telomeric Protein

Abstract G-quadruplexes are four-stranded nucleic acid structures involved in multiple cellular pathways including DNA replication and telomere maintenance. Such structures are formed by G-rich DNA sequences typified by telomeric DNA repeats. Whilst there is evidence for proteins that bind and regulate G-quadruplex formation, the molecular basis for this remains poorly understood. The budding yeast telomeric protein Rap1, originally identified as a transcriptional regulator functioning by recognizing double-stranded DNA binding sites, was one of the first proteins to be discovered to also bind and promote G-quadruplex formation in vitro. Here, we present the 2.4 Å resolution crystal structure of the Rap1 DNA-binding domain in complex with a G-quadruplex. Our structure not only provides a detailed insight into the structural basis for G-quadruplex recognition by a protein, but also gives a mechanistic understanding of how the same DNA-binding domain adapts to specifically recognize different DNA structures. The key observation is the DNA-recognition helix functions in a bimodal manner: In double-stranded DNA recognition one helix face makes electrostatic interactions with the major groove of DNA, whereas in G-quadruplex recognition a different helix face is used to make primarily hydrophobic interactions with the planar face of a G-tetrad.

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The DNA-binding domain of BenM reveals the structural basis for the recognition of a T-N11-A sequence motif by LysR-type transcriptional regulators

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444913017320 ◽

2013 ◽

Vol 69 (10) ◽

pp. 1995-2007 ◽

Cited By ~ 27

Author(s):

Amer M. Alanazi ◽

Ellen L. Neidle ◽

Cory Momany

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Dna Recognition ◽

Transcriptional Regulators ◽

Binding Domain ◽

Structural Basis ◽

Binding Motif ◽

Sequence Motif ◽

Dna Binding Domain ◽

Half Site

LysR-type transcriptional regulators (LTTRs) play critical roles in metabolism and constitute the largest family of bacterial regulators. To understand protein–DNA interactions, atomic structures of the DNA-binding domain and linker-helix regions of a prototypical LTTR, BenM, were determined by X-ray crystallography. BenM structures with and without bound DNA reveal a set of highly conserved amino acids that interact directly with DNA bases. At the N-terminal end of the recognition helix (α3) of a winged-helix–turn–helix DNA-binding motif, several residues create hydrophobic pockets (Pro30, Pro31 and Ser33). These pockets interact with the methyl groups of two thymines in the DNA-recognition motif and its complementary strand, T-N11-A. This motif usually includes some dyad symmetry, as exemplified by a sequence that binds two subunits of a BenM tetramer (ATAC-N7-GTAT). Gln29 forms hydrogen bonds to adenine in the first position of the recognition half-site (ATAC). Another hydrophobic pocket defined by Ala28, Pro30 and Pro31 interacts with the methyl group of thymine, complementary to the base at the third position of the half-site. Arg34 interacts with the complementary base of the 3′ position. Arg53, in the wing, provides AT-tract recognition in the minor groove. For DNA recognition, LTTRs use highly conserved interactions between amino acids and nucleotide bases as well as numerous less-conserved secondary interactions.

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