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

Abstract

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 ...

In vivo and in vitro studies of Chlamydia trachomatis TrpR:DNA interactions

“…The ability to protect the operator in vitro and repress trpBA expression in vivo is a function of the affinity of TrpR for the various corepressors and/or the affinity of the TrpR/corepressor complex for the operator DNA sequence. In E. coli , it has been shown that 5‐fluorotryptophan binds with similar affinity and in the same orientation as l ‐tryptophan, and is an equally effective corepressor (Borden et al ., 1991). In addition, different tryptophan analogues have varying affinities for purified TrpR and the subsequent analogue–TrpR complexes show differential binding affinities for the E. coli operator sequence (Marmorstein and Sigler, 1989).…”

A negative electrostatic determinant mediates the association between the Escherichia coli trp repressor and its operator DNA

“…Some tryptophan analogues bind more tightly to the aporepressor than does tryptophan, induce the conformational change, and yet these form protein-ligand complexes or pseudorepressors that cannot bind to DNA (Marmorstein et al, 1987;Marmorstein & Sigler, 1989). For example, the tryptophan analogue, indole-3-propionate (IPA), lacks the positively charged amino group found on tryptophan and binds to the protein using a different binding mode than tryptophan Borden et al, 1991). Lawson and co-workers proposed that the IPA binding mode would interfere with pseudorepressor-DNA binding due to steric and electrostatic clashes between the IPA carboxylate and a DNA backbone phosphate.…”

Interaction of the trp repressor with trp operator DNA fragments

“…We have previously reported that the mobility of the 1 : 1 trp holorepressor: trpO complex is not dependent on the nature of the corepressor (Borden 1991). However, the different apparent sizes of the various complexes that are formed are dependent on the nature of the DNA target, particularly the 1 : 1 complexes or the holorepressor with trpO and aroH.…”

“…Trp aporepressor was purified from an overproducing mutant as previously described (Joachimiak et al 1983, Borden et al 1991. The protein was > 95% pure judging from SDS electrophoresis.…”