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 ...

Sequence-specific I5N and 'H assignments for the trp holorepressor from Escherichia coli are reported. The trp repressor consists of two identical 107-residue subunits which are highly helical in the crystal state [Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L. & Sigler, P. B. (1985) Nature 31 7, 782 -7861. The high helical content and the relatively large size of the protein (Mr = 25 000) make it difficult to assign even the main-chain resonances by conventional homonuclear twodimensional NMR methods. However, we have now assigned the main-chain resonances of 94% of the residues by using three-dimensional ' 5N/'H heteronuclear experiments on a sample of protein uniformly labelled with "N. The additional resolution obtained by spreading out the signals into three dimensions proved indispensable in making these assignments. In particular, we have been able to resolve signals from residues in the N-terminal region of the A helix for the first time in solution. The observed NOE results confirm that the repressor is highly helical in solution, and contains no extended chain conformations.The trp repressor is a 25-kDa dimeric protein which controls transcription of the trpO, trpR and aroH operons in Escherirhiu coli (Platt, 1980;Klig et al., 1988). The trp repressor regulates transcription by selectively binding to a 20-base-pair operator and interfering with RNA polymerase activity. The DNA binding affinity is allosterically regulated by intracellular levels of L-tryptophan (It0 and Yanofsky, 1969).The structure of the trp repressor has been extensively studied crystallographically by Sigler and coworkers (Schevitz et al., 1985;Zhang et al., 1987;Lawson et al., 1988;Otwinowski et al., 1988). The trp repressor dimer has 107 residues/subunit ; each subunit consists of six helices separated by /j' turns. The DNA-binding domain is formed by the D and E helices which comprise the helix-turn-helix motif common to many prokaryotic and phage DNA-binding proteins (for reviews see Steitz, 1990;Freemont et al., 1991). NMR studies to date have confirmed that the protein consists of six helices in solution and that the N-terminal arm is extremely flexible (Arrowsmith et al., 1990a, b;Lane, 1989). It has been postulated that this N-terminal arm may be involved in stabilising the DNA repressor complex by wrapping around the DNA as in the 1, repressors (Carey, 1989). More recently, this hypothesis has been questioned based on measurements of the effect of removing the first six residues on the affinity for operator DNA (Marmorstein et al., 1991).One of the first steps in probing the nature of the trprepressor -DNA interaction by NMR is the assignment of the _ _ _ _

The interaction of the trp repressor with several trp operator DNA fragments has been examined by DNA gel retardation assays and by circular dichroism, in the absence and presence of the corepressor L-tryptophan. The holorepressor binds stoichiometrically to both the trpO and aroH operators, forming 1:1 complexes. In the presence of excess protein, additional complexes are formed with these operator fragments. The relative electrophoretic mobilities of the 1:1 complexes differ significantly for trp and aroH operators, indicating that they differ substantially in gross structure. A mutant trp operator, trpOc, has low affinity for the holorepressor, and forms only complexes with stoichiometries of 2:1 (repressor: DNA) or higher, which have a very low electrophoretic mobility. Specific binding is also accompanied by a large increase in the intensity of the near ultraviolet circular dichroism, with only a small blue shift, which is consistent with significant changes in the conformation of the DNA. Large changes in the chemical shifts of three resonances in the 31P NMR spectrum of both the trp operator and the aroH operator occur on adding repressor only in the presence of L-tryptophan, consistent with localised changes in the backbone conformation of the DNA.