Determination of the orientations of tryptophan analogues bound to the trp repressor and the relationship to activation.
The antirepressor indole 3-propanoate has been shown by X-ray crystallography to bind in a different orientation compared with the natural corepressor for the tryp repressor, L-tryptophan (Lawson, C.L. & Sigler, P. B. (1988) Nature 333, 869-871). This suggests a simple difference between what constitutes a corepressor versus an antirepressor. We have used visible absorption and 1H-NMR spectroscopy to characterise the nature of several ligand-repressor 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 313 K. These findings complement and extend the X-ray and thermodynamic analyses of ligand binding.
Borden
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KL
National Institute for Medical Research, Mill Hill, London, England.
Beckmann
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Lane
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D001426:Bacterial Proteins; D004247:DNA / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism*; D014961:X-Ray Diffraction; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D004247:DNA; D007211:Indoles; D008024:Ligands; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins; C015292:indolepropionic acid