Analysis of the conformation and stability of rat TTF‐1 homeodomain by circular dichroism

Damante, Giuseppe; Tella, Gianluca; Leonardi, Antonio; Fogolari, Federico; Bortolotti, Nadia; Lauroa, Roberto Di; Formisano, Silvestro

doi:10.1016/0014-5793(94)01145-1

Cited by 22 publications

(27 citation statements)

References 25 publications

(13 reference statements)

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“…1a), and similar to that observed for other homeodomains (21,30,31). A secondary structure prediction algorithm applied to the spectra yielded an ␣-helical content of 41% (Fig.…”

Section: Resultssupporting

confidence: 79%

“…The T m (44°C) is comparable with that of other known homeodomains (30,31). The conservative mutation W48F, designed to minimally perturb the overall hydrophobicity of the helix III environment, has a significant ␣-helical structure yet exhibits 50-fold lower affinity for DNA, suggesting that Trp-48 plays a more complex role in DNA binding than that postulated previously.…”

Section: An Aromatic Residue In Position 8 Is Required For Stability mentioning

confidence: 62%

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Aromatic Amino Acids Are Critical for Stability of the Bicoid Homeodomain

Subramaniam¹,

Jovin

Rivera‐Pomar

2001

Journal of Biological Chemistry

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The Drosophila Bicoid (Bcd) protein plays a dual role as a transcription and translation factor dependent on the unique DNA and RNA binding properties of the homeodomain (HD). We have used circular dichroism and fluorescence spectroscopy to probe the structure and stability of the Bcd-HD, for which a high resolution structure is not yet available. The fluorescence from the single tryptophan residue in the HD (Trp-48) is strongly quenched in the native state but is dramatically enhanced (ϳ20-fold) upon denaturation. Similar results were obtained with the Ultrabithorax HD (Ubx-HD), suggesting that the unusual tryptophan fluorescence may be a general phenomenon of HD proteins. We have used site-directed mutagenesis to explore the role of aromatic acids in the structure of the Bcd-HD and to evaluate the proposal that interactions between the strictly conserved Trp residue in HDs and nearby aromatic residues are responsible for the fluorescence quenching in the native state. We determined that both Trp-48 and Phe-8 in the N-terminal region of the HD are individually necessary for structural stability of the Bcd-HD, the latter most likely as a factor coordinating the orientation of the N-terminal helix I and the recognition helix for efficient binding to a DNA target.The regulation of gene expression is generally accomplished by interactions of proteins with nucleic acids. These regulators contain DNA binding domains (transcription factors) or RNA binding domains (translational regulators). The homeobox encodes a 60-amino acid nucleic acid binding domain, the homeodomain (HD), 1 which is present in many eukaryotic transcription factors and plays a critical role in development (1, 2). The sequences have been well conserved in the course of evolution from fungi to vertebrates. In addition to binding DNA, thereby activating transcription, the Drosophila Bcd-HD has been recently shown to interact with RNA (3-5). Thus, Bcd has a dual role in development, directing pattern formation along the anterior-posterior axis of the embryo by cooperative DNA binding and subsequent gene activation (6 -8) and by binding the mRNA of the regulatory gene caudal (cad), thereby repressing its translation (3, 5).Because of this unique DNA and RNA binding capability, Bcd-HD is a particularly compelling target for investigations of molecular structure and nucleic acid recognition mechanisms.The HD has also become a paradigm for studying DNAprotein interactions. HDs fold into a module with a characteristic structure comprising three ␣-helices. Structural studies, first elucidated for the Antennapedia and Engrailed HDs, show that the three helices of the HD are stabilized by hydrophobic interactions involving residues Leu-16, Trp-48, and Phe-49, which are conserved in all HDs (9 -15). The HD shows a common helix-turn-helix motif encompassing the recognition helix III, which docks into the major groove of DNA. The specificity of DNA recognition is achieved by specific amino acid residuebase contacts in helix III (16), e.g. amino acid residue 50 (...

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“…1a), and similar to that observed for other homeodomains (21,30,31). A secondary structure prediction algorithm applied to the spectra yielded an ␣-helical content of 41% (Fig.…”

Section: Resultssupporting

confidence: 79%

Section: An Aromatic Residue In Position 8 Is Required For Stability mentioning

confidence: 62%

Aromatic Amino Acids Are Critical for Stability of the Bicoid Homeodomain

Subramaniam¹,

Jovin

Rivera‐Pomar

2001

Journal of Biological Chemistry

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“…H-D-exchange and thermal-denaturation data (Damante et al, 1994b) suggest that some regions of the molecule are less rigid than others under the chosen experimental conditions, which inherently conflicts with a precise conformational definition. Whilst increased local mobility could account for lower-distance-bound violations (especially if one considers that a single calibrant connectivity from a rather rigid region was used for NOE quantitation), underestimation of upper limits could arise from limited spin diffusion, which conceivably affected the employed NOE-amplitude data set.…”

Section: Nmr Assignments and Secondary-structure Identificationmentioning

confidence: 97%

“…Its melting temperature, as determined by CD thermal-denaturation studies (Damante et al, 1994b), i s 314 K, and with an estimated of -5.9 kJ/ mol at 298 K, a significant loss of structure is expected at room temperature. Whilst thermal lability could have been anticipated from early NMR data, the choice of the experimental temperature range for structural determinations was dictated by qualitative inspection of the NOESY spectra.…”

Section: Nmr Assignments and Secondary-structure Identificationmentioning

confidence: 99%

“…A homology-modeled structure of the isolated domain and of its DNA complex was proposed , which confirmed the available functional evidence and predicted a specific role for rcsidue 54. The secondary structure of TTF-1 homeodomain was determined by 'H-NMR , and comparative electrophoretic-mobilityshift-assay and CD studies were performed to assess a variety of thermodynamic-stability and binding parameters (Damante et al, , 1994b.…”

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Analysis of the Solution Structure of the Homeodomain of Rat Thyroid Transcription Factor 1 by ¹H‐NMR Spectroscopy and Restrained Molecular Mechanics

Esposito

Fogolari

Damante

et al. 1996

European Journal of Biochemistry

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The solution structure of the rat thyroid transcription factor 1 (TTF-1) homeodomain has been elucidated by 'H-NMR and restrained modeling. The TTF-1 homeodomain folds in the same manner as classical homeodomains, with three helices, a loose loop between the first two helices, and a tight turn between helix I1 and helix 111. The typical assembly of the hydrophobic core is maintained and N-capping motifs are identified in helix I and helix 111. The N-terminal stretch of helix IT exhibits some mobility, similar to the preceding loop region, which may be related to its anomalous capping. The N-terminal decapeptide and the C-terminal octapeptide of the molecule (68 residues long) are disordered. All the previous characteristics are shared by all known isolated homeodomain structures. An important difference among these structures occurs at the C-terminal extension of helix 111, which is either disordered or helically folded. In the TTF-1 homeodomain, the C-terminal extension of helix Ill (residues 51 -59) appears structured, albeit not as rigidly as the preceding portion. Analysis of the NOES and hydrogendeuterium exchange of backbone amides provides evidence for discontinuity between the two moieties of helix 111, which is introduced by a tightening or a kink of residues 51 -53.

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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 α‐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–π 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–π interactions that connect secondary structures. © 2001 John Wiley & Sons, Inc. Biopolymers 59: 145–159, 2001

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Analysis of the conformation and stability of rat TTF‐1 homeodomain by circular dichroism

Cited by 22 publications

References 25 publications

Aromatic Amino Acids Are Critical for Stability of the Bicoid Homeodomain

Aromatic Amino Acids Are Critical for Stability of the Bicoid Homeodomain

Analysis of the Solution Structure of the Homeodomain of Rat Thyroid Transcription Factor 1 by ¹H‐NMR Spectroscopy and Restrained Molecular Mechanics

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

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Analysis of the conformation and stability of rat TTF‐1 homeodomain by circular dichroism

Cited by 22 publications

References 25 publications

Aromatic Amino Acids Are Critical for Stability of the Bicoid Homeodomain

Aromatic Amino Acids Are Critical for Stability of the Bicoid Homeodomain

Analysis of the Solution Structure of the Homeodomain of Rat Thyroid Transcription Factor 1 by 1H‐NMR Spectroscopy and Restrained Molecular Mechanics

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

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Analysis of the Solution Structure of the Homeodomain of Rat Thyroid Transcription Factor 1 by ¹H‐NMR Spectroscopy and Restrained Molecular Mechanics