The three-dimensional structure of trp repressor

Schevitz, Richard W.; Otwinowski, Zbyszek; Joachimiak, A.; Lawson, Catherine L.; Sigler, Paul B.

doi:10.1038/317782a0

Cited by 355 publications

(229 citation statements)

References 28 publications

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“…It may be that the tryptophan orientation is intrinsically less favoured than the indolepropanoate orientation, and can only be fully populated when the ammonium group is able to make appropriate interactions with the protein. This is in line with the observation that most of the binding energy derives from the indole ring of the ligand [2], and that the side chain of L-tryptophan is in an unusual conformation in the bound state [24]. Further, in the indolepropanoate orientation, the indole NH makes a good hydrogen bond with the peptide oxygen of Leu41 [3], whereas in the tryptophan orientation, no hydrogen bond can form to the protein as the NH points out toward the solvent [3,4,25].…”

Section: Orirntution Oj'bound Ligundsupporting

confidence: 71%

Determination of the orientations of tryptophan analogues bound to the trp repressor and the relationship to activation

Borden

Beckmann

Lane

1991

European Journal of Biochemistry

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The antirepressor indole 3-propanoate has been shown by X-ray crystallography to bind in a different We have used visible absorption and 'H-NMR spectroscopy to characterise the nature of several ligandrepressor complexes and DNA-binding assays to assess the relative operator binding affinities. 5-Fluorotryptophan binds with similar affinity and in the same orientation as L-tryptophan, and is an equally effective corepressor. In contrast, the tight-binding antirepressor indole 3-acrylate binds in the same orientation as indole 3-propanoate. Indole, also an antirepressor, also binds in the indole-3-propanoate orientation. 5-Methyltryptamine, a corepressor, shows spectroscopic characteristics of both tryptophan and indoleacrylate, though NOEs indicate that the tryptophan orientation is preferred. These results indicate that the ammonium group in the side chain is essential both for activation and binding in the L-tryptophan orientation. Antirepressors, lacking the ammonium group, bind in the more favourable indole-3-propanoate orientation.Differences in the NMR signatures of the different repressor-ligand complexes indicate that the details of the conformations depend on the nature of the ligands and their orientation within the binding site. Despite any conformational rearrangement of the protein on binding, dissociation of ligands is facile: 5-fluorotryptophan dissociates rapidly at 31 3 K. These findings complement and extend the X-ray and thermodynamic analyses of ligand binding.The expression of the tryptophan biosynthetic enzymes is controlled by positive repression by the end product of the pathway, L-tryptophan. The trp aporepressor has only weak, nonspecific affinity for DNA [l], but on binding the corepressor, L-tryptophan, the protein is activated and binds the trp operator with high affinity.Extensive ligand-binding studies have shown that, while a wide range of indole-based molecules bind to the trp repressor with high affinity, only those containing the ammonium group on the side chain are able to activate the repressor [2]. Further, Sigler and coworkers [3] have shown that the antirepressor indole 3-propanoate binds in a different orientation to tryptophan, and is rotated about 180" such that the indole NH points toward the interior of the protein rather than toward the solvent (and the DNA backbone) [4]. This finding has led to the proposal that the necessary requirements for forming a functional repressor are the indole ring (which provides most of the binding energy), the ammonium group and the indole NH that forms a hydrogen bond to the DNA. Hence, in order to activate the repressor, the ligand must bind in the correct orientation. One intriguing finding, however, is that D-tryptophan, while having only low affinity for the repressor, is a more effective corepressor than the L-isomer [ 5 ] . To test the hypothesis that activation requires a particular orientation of the bound ligand within the binding pocket, it is important to determine the relative orientation of different ligands, and their ...

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Section: Orirntution Oj'bound Ligundsupporting

confidence: 71%

Determination of the orientations of tryptophan analogues bound to the trp repressor and the relationship to activation

Borden

Beckmann

Lane

1991

European Journal of Biochemistry

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“…The crystal structures of the holorepressor and aporepressor show that the subunits contain six a-helices, two of which correspond to the helix-turn-helix structure as observed in other prokaryotic repressors, and an N-terminal arm of eleven residues (Schevitz et al, 1985;Zhang et al, 1987 (1988).…”

Section: Helix-turn-helix Proteinsmentioning

confidence: 94%

Structural aspects of protein-DNA recognition

Freemont

Lane

Sanderson

1991

Biochemical Journal

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“…Its structure in both the Trp-free apo-form and the Trp-bound holo-form, and the structure of its DNA complexes, have been determined by both X-ray and NMR analyses (Schevitz et al, 1985;Otwinowski et al, 1988;Lawson & Carey, 1993;Zhao et al, 1993;Zhang et al, 1994). The repressor is known to interact with at least five different operator sequences, i.e., in the trp, trpR, aroH, aroL, and mtr operons Reprint requests to: Oleg Jardetzky, Stanford Magnetic Resonance Laboratory, Stanford University, Stanford, California 94305.5055; e-mail: jardetzky@camis.stanford.edu.…”

Section: " " ~~~mentioning

confidence: 99%

Flexibility of dna binding domain of trp repressor required for recognition of different operator sequences

et al. 1996

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Trp repressor (25 kDa) is a regulatory protein that controls transcription initiation in the tryptophan biosynthetic operon and at least four other operons in Escherichia coli. An alanine to valine mutation (AV77) in the DNA binding domain is known to increase repressor activity at the trp operator in vivo, but not in vitro. We report here the amide proton exchange rates for the DNA-binding domains of both the wild-type and AV77 proteins. We find that the alanine to valine change stabilizes the flexible DNA-binding domain of the repressor. We present in vivo data showing that, although the AV77 repressor is more inhibitory at the trp operator than the wild-type repressor, it does not have increased activity at the aroH or trpR operator; repression at the aroH operator is, in fact, reduced. Our results suggest that the flexibility exhibited by the wild-type repressor allows a broader range of repressor/DNA interactions, whereas the increased rigidity resulting from the AV77 change limits the repressor's effectiveness at some operators.

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The three-dimensional structure of trp repressor

Cited by 355 publications

References 28 publications

Determination of the orientations of tryptophan analogues bound to the trp repressor and the relationship to activation

Determination of the orientations of tryptophan analogues bound to the trp repressor and the relationship to activation

Structural aspects of protein-DNA recognition

Flexibility of dna binding domain of trp repressor required for recognition of different operator sequences

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