Effects of DNA binding and metal substitution on the dynamics of the GAL4 DNA‐binding domain as studied by amide proton exchange

Mau, Ted; Baleja, James D.; Wagner, Gerhard

doi:10.1002/pro.5560011102

Cited by 18 publications

(6 citation statements)

References 30 publications

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“…Cadmium ions are significantly larger than zinc ions, thus it would seem likely that the protein structure would alter in some manner to accommodate the larger cadmium ions. Indeed, 2D NMR (Gardner et al, 1991) and amide proton exchange results (Mau et al, 1992) are indicative of a more open structure for the Cd2 form of the protein. Despite these changes, the Cd for Zn substitution results in relatively modest changes in the affinity of the protein for its specific DNA sequence, 2-3-fold, compared to the = 103-fold preference for specific vs. nonspecific DNA under these gel conditions (Fig.…”

Section: Discussionmentioning

confidence: 99%

DNA binding and bending by the transcription factors GAL4(62) and GAL4(149)

Rodgers

Coleman

1994

Protein Science

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The DNA binding domain of the GAL4 transcription factor from yeast is located in the N-terminal60 residues of the polypeptide of 881 amino acids. This domain binds 2 Zn ions, which form a binuclear cluster, ZnzC,, with 6 C residues, two of which bridge the 2 metal ions (Gardner KH et al., 1991, Biochemistry 3 0 11292-1 1302). Binding of Zn or Cd to GAL4 induces the conformation of the protein necessary to recognize the specific DNA sequence, UASG, to which GAL4 binds as a dimer. Gel retardation assays have been utilized to determine the relative affinities of the Zn, and Zn, forms of the N-terminal 149 residues of GAL4, GAL4(149*), for UAS, DNA sequences. We show that Cd2-and ZnlGAL4(149*) bind to UASG DNA with 2-fold and 4-8-fold lower affinities than ZnzGAL4(149*), respectively. Thus, the metal species and the number of metal ions bound have measurable effects on the specific DNA binding affinity of GAL4, but these differences are small in comparison to the ratio, >lo3 under some conditions, that characterizes the specific to nonspecific DNA binding affinities of the N-terminal fragments of GAL4. A shorter N-terminal fragment, GAL4(62*), although it continues to recognize the UASG sequence with a high degree of specificity, binds with 1,000-2,000-fold lower affinity than does ZnzGAL4(149*). Gel retardation titrations of a DNA containing 2 UASG sites with increasing concentrations of GAL4(62*) generate a series of 4 retarded bands in contrast to 2 retarded bands formed when the the same DNA is titrated with GAL4( 149*). These data suggest that GAL4(62*) binds to the UASG sites as individual monomers that dimerize on the DNA, whereas GAL4(149*) binds the UASG DNA cooperatively as a dimer. The =IO3 lower affinity of GAL4(62*) for the UASG DNA can be accounted for by its failure to form dimers in solution. Zn,-, Znl-, or Cd2GAL4(149*) induces differential rates of gel migration in a series of circularly permutated UASGcontaining DNA restriction fragments. Analysis of the data suggests that all 3 proteins cause a 26" angle of bend in the DNA when bound to 1 UASG site and 45" when bound to 2 tandem UASG sites. The same assay shows that GAL4(62*) does not induce significant bending of the UAS, DNA sequences. Thus, the additional subdomains found in the larger polypeptide fragment, GAL4(149*), must exert an additional force on the DNA either through direct contacts with the DNA or indirectly through altered protein conformation.

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Section: Discussionmentioning

confidence: 99%

DNA binding and bending by the transcription factors GAL4(62) and GAL4(149)

Rodgers

Coleman

1994

Protein Science

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“…It is exactly this point that calls into question the notion that allosteric pathways are precisely conserved across a family. The literature includes nearly countless examples demonstrating that protein structures and their dynamics are highly sensitive to small perturbations, regardless of whether the perturbation is mutation (53), ligand binding (54), or simply changing the type of metal ion bound to the protein (55). Related, several reports stress the diversity within dynamic signatures across protein families (56,57).…”

Section: A Critical View Of the Underlying Concept Of Conserved Allosmentioning

confidence: 99%

A Critical Evaluation of Correlated Mutation Algorithms and Coevolution Within Allosteric Mechanisms

Livesay

Kreth

Fodor

2011

Methods in Molecular Biology

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The notion of using the evolutionary history encoded within multiple sequence alignments to predict allosteric mechanisms is appealing. In this approach, correlated mutations are expected to reflect coordinated changes that maintain intramolecular coupling between residue pairs. Despite much early fanfare, the general suitability of correlated mutations to predict allosteric couplings has not yet been established. Lack of progress along these lines has been hindered by several algorithmic limitations including phylogenetic artifacts within alignments masking true covariance and the computational intractability of consideration of more than two correlated residues at a time. Recent progress in algorithm development, however, has been substantial with a new generation of correlated mutation algorithms that have made fundamental progress toward solving these difficult problems. Despite these encouraging results, there remains little evidence to suggest that the evolutionary constraints acting on allosteric couplings are sufficient to be recovered from multiple sequence alignments. In this review, we argue that due to the exquisite sensitivity of protein dynamics, and hence that of allosteric mechanisms, the latter vary widely within protein families. If it turns out to be generally true that even very similar homologs display a wide divergence of allosteric mechanisms, then even a perfect correlated mutation algorithm could not be reliably used as a general mechanism for discovery of allosteric pathways.

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“…One should clearly keep in mind that the present analysis was performed on the biologically significant Zn complex and not on the Cd complex. Mau et al [28] have showed that some amide protons exchange 3–21× faster in Cd‐GAL4 than in Zn‐GAL4 and suggested that cadmium substitution is likely to cause a reduction of the global stability of the protein and of the recognition module. Rodgers et al [29] showed that the Zn for Cd substitution results in relatively modest changes (2–3‐fold decrease) in the affinity of a fragment of GAL4, comprising both the DNA‐binding domain and an additional subdomain (GAL4(149 * )), for its specific DNA sequence.…”

Section: Discussionmentioning

confidence: 99%

The Aspergillus nidulans transcription factor AlcR forms a stable complex with its half‐site DNA: a NMR study

Cerdan¹,

Collin²,

Lenouvel

et al. 1997

FEBS Letters

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The Aspergillus nidulans transcription factor AlcR is shown by NMR and gel retardation assay to form a stable complex with oligonucleotide sequences comprising the consensus half-site 5'-TGCGG-3'. Apparent /jM dissociation constants are evaluated by both methods. The measured lifetime of the complex is 74 ±7 ms at 20°C with the following DNA sequence: 5'-C1G2T3G4C5G6G7A8T9C10-3'. The major chemical shift variations upon binding involve both the two adjacent GC pairs (G6 and G7) and, clearly, the AT pairs at both ends of the consensus sequence (T3 and A8), suggesting additional contacts of the protein with the DNA. This extensive and strong interaction with the half-site is another example of the variability in contacts of the fungal DNA-binding proteins containing Zn 2 Cys 6 domains with their consensus sites. It is the first demonstration that a binuclear cluster protein can bind to DNA as a monomer with strong affinity.

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Effects of DNA binding and metal substitution on the dynamics of the GAL4 DNA‐binding domain as studied by amide proton exchange

Cited by 18 publications

References 30 publications

DNA binding and bending by the transcription factors GAL4(62) and GAL4(149)

DNA binding and bending by the transcription factors GAL4(62) and GAL4(149)

A Critical Evaluation of Correlated Mutation Algorithms and Coevolution Within Allosteric Mechanisms

The Aspergillus nidulans transcription factor AlcR forms a stable complex with its half‐site DNA: a NMR study

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Effects of DNA binding and metal substitution on the dynamics of the GAL4 DNA‐binding domain as studied by amide proton exchange

Cited by 18 publications

References 30 publications

DNA binding and bending by the transcription factors GAL4(62*) and GAL4(149*)

DNA binding and bending by the transcription factors GAL4(62*) and GAL4(149*)

A Critical Evaluation of Correlated Mutation Algorithms and Coevolution Within Allosteric Mechanisms

The Aspergillus nidulans transcription factor AlcR forms a stable complex with its half‐site DNA: a NMR study

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DNA binding and bending by the transcription factors GAL4(62) and GAL4(149)

DNA binding and bending by the transcription factors GAL4(62) and GAL4(149)