Electrostatics and hydration at thehomeodomain-DNA interface: chemical probes of an interfacial water cavity

Labeots, Laura A.; Weiss, Michael A.

doi:10.1006/jmbi.1997.1026

Cited by 19 publications

(20 citation statements)

References 82 publications

(144 reference statements)

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“…Our approach exploited the site-specific substitution of the negatively charged phosphodiester linkage by a neutral analog (methylphosphonate). Such modifications can interfere with protein binding and so yield a discrete footprint (10,44).…”

Section: Discussionmentioning

confidence: 99%

“…Methylphosphonate interference (10, 53) was evaluated by gel mobility-shift assay (GMSA) in a 29-bp DNA duplex (5Ј-GTGCACAACTACAATGTTGCAATCAGCGG-3Ј and its complement; bold indicates the dsx A site). Possible stereospecific interference at selected sites was evaluated following high-performance liquid chromatography (HPLC) resolution of single-strand diastereomers (44). DNA-binding reactions were conducted in 20 mM Tris-HCl (pH 7.4), 150 mM KCl, 5 mM MgCl 2 , 0.1 mM ZnCl 2 , 5% glycerol, 33 g/ml bovine serum albumin, and 0.08 g/l poly(dI-dC) competitor DNA.…”

Section: Methodsmentioning

confidence: 99%

“…Chemical evidence for the function of one such site, a conserved lysine on the surface of the Zn module (K60) (Fig. 1D, arrow), is obtained by an in vitro complementation test: replacement of a putative salt bridge to the DNA backbone by a novel "hydrophobic bridge" between a variant domain and a nonstandard DNA analog containing a neutral phosphodiester modifi- cation (10,44). Together, the dual use of mutational and chemogenetic approaches suggests a general strategy for the characterization of nucleic-acid-binding domains.…”

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confidence: 99%

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Regulation of Sexual Dimorphism: Mutational and Chemogenetic Analysis of the Doublesex DM Domain

Zhang

Singh

et al. 2006

Molecular and Cellular Biology

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Doublesex (dsx) is a transcription factor in Drosophila that regulates somatic sexual differentiation. Maleand female-specific splicing isoforms of DSX share a novel DNA-binding domain, designated the DM motif. Broadly conserved among metazoan sex-determining factors, the DM domain contains a nonclassical zinc module and binds in the DNA minor groove. Here, we characterize the DM motif by site-directed and random mutagenesis using a yeast one-hybrid (Y1H) system and extend this analysis by chemogenetic complementation in vitro. The Y1H system is based on a sex-specific Drosophila enhancer element and validated through studies of intersexual dsx mutations. We demonstrate that the eight motif-specific histidines and cysteines engaged in zinc coordination are each critical and cannot be interchanged; folding also requires conserved aliphatic side chains in the hydrophobic core. Mutations that impair DNA binding tend to occur at conserved positions, whereas neutral substitutions occur at nonconserved sites. Evidence for a specific salt bridge between a conserved lysine and the DNA backbone is obtained through the synthesis of nonstandard protein and DNA analogs. Together, these results provide molecular links between the structure of the DM domain and its function in the regulation of sexual dimorphism.Sexual dimorphism is regulated by diverse molecular mechanisms (18). In mammals, male development is initiated by a gene on the Y chromosome that is designated Sry (sex-determining region of the Y chromosome) (7,30,41,42). Alternative genetic mechanisms operate in Drosophila melanogaster (and Caenorhabditis elegans), wherein sex is determined by the X-to-autosome ratio, a process linked to X-dosage compensation (Fig. 1A) (19, 59). The sex-determining hierarchy of Drosophila defines an RNA-splicing cascade through the actions of Sex Lethal (SXL) and the TRA factors (18). Doublesex (dsx) functions downstream of this cascade to define one branch of a ramifying pathway (Fig. 1A) (29,59). The sex-specific isoforms of DSX (DSX M and DSX F ; Fig. 1B) regulate most aspects of somatic sexual dimorphism in D. melanogaster. These isoforms share an N-terminal DNA-binding domain, designated the DM motif (Fig. 1C). In this article, we present a mutational analysis of the DSX DM domain to identify key determinants of folding and DNA recognition.The Wilkins-Wolf hypothesis posits that sex-determining pathways have evolved in a reverse order from bottom (most broadly conserved) to top (least conserved) (65, 66). This viewpoint is supported by the ubiquity of DM genes among metazoan sex-determining pathways (35, 59). Such genes encode a newly recognized class of transcription factors (for a review, see reference 59). DM sequences are broadly conserved in metazoans, including nematodes, fish, reptiles, birds, and mammals (Fig. 1D). The deletion of human DM genes on chromosome 9p is associated with intersexual development (6,27,31,49,55,59,63). Although originally described in relation to sex-determining pathways (67), an extended family...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Regulation of Sexual Dimorphism: Mutational and Chemogenetic Analysis of the Doublesex DM Domain

Zhang

Singh

et al. 2006

Molecular and Cellular Biology

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“…63 The modified DNA probes are ordinarily racemic mixtures of R p and S p diastereomers. 64 Classical binding of globular domains to one face of B-DNA yields characteristic patterns of interference in the major or minor grooves (Figure 6(a) and (b)). By contrast, the structure of the SRY-DNA complex 42 predicts a non-classical pattern of interference (filled circles below).…”

Section: Methylphosphonate Interference Yields Similar Footprintsmentioning

confidence: 98%

SRY and Human Sex Determination: The Basic Tail of the HMG Box Functions as a Kinetic Clamp to Augment DNA Bending

Phillips

Jancso-Radek

Ittah

et al. 2006

Journal of Molecular Biology

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“…They all consist of three ␣-helices, preceded by an N-terminal arm ( Figure 1). 12 Specificity is finally increased through the association with other monomers or proteins that induce cooperative DNA binding. The second and third helices constitute the typical HTH motif, whereas the position of the first helix is specific to the homeodomain subfamily.…”

Section: Introductionmentioning

confidence: 99%

Role of salt bridges in homeodomains investigated by structural analyses and molecular dynamics simulations

et al. 2001

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Homeodomains are a class of helix-turn-helix DNA-binding protein motifs that play an important role in the control of cellular development in eukaryotes. They fold in a three alpha-helix structural module, where the third helix is the recognition helix that fits into the major groove of DNA. Structural analysis of the members of the homeodomain family led to the identification of interactions likely to stabilize the protein domains. Linking the helices pairwise, three salt bridges were found to be well preserved within the family. Also well conserved were two cation-pi interactions between aromatic and positively charged side chains. To analyze the structural role of the salt bridges, molecular dynamics simulations (MD) were carried out on the wild-type homeodomain from the Drosophila paired protein (1fjl) and on three mutants, which lack one or two salt bridges and mimic natural mutations in other homeodomains. Analysis of the trajectories revealed only small structural rearrangements of the three helices in all MD simulations, thereby suggesting that the salt bridges have no essential stabilizing role at room temperature, but rather might be important for improving thermostability. The latter hypothesis is supported by a good correlation between the melting midpoint temperatures of several homeodomains and the number of salt bridges and cation-pi interactions that connect secondary structures.

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Electrostatics and hydration at thehomeodomain-DNA interface: chemical probes of an interfacial water cavity

Cited by 19 publications

References 82 publications

Regulation of Sexual Dimorphism: Mutational and Chemogenetic Analysis of the Doublesex DM Domain

Regulation of Sexual Dimorphism: Mutational and Chemogenetic Analysis of the Doublesex DM Domain

SRY and Human Sex Determination: The Basic Tail of the HMG Box Functions as a Kinetic Clamp to Augment DNA Bending

Role of salt bridges in homeodomains investigated by structural analyses and molecular dynamics simulations

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