NMR Studies of the Escherichia coli Trp Repressor ·trpR^s Operator Complex

Abstract

To understand the specificity of the Escherichia coli Trp repressor for its operators, we have begun to study complexes of the protein with alternative DNA sequences, using 'H-NMR spectroscopy. We report here the 'H-NMR chemical shifts of a 20-bp oligodeoxynucleotide containing the sequence of a symmetrised form of the trpR operator in the presence and absence of the holorepressor. Deuterated protein was used to assign the spectrum of the oligodeoxynucleotide in a 37-kDa complex with the Trp holorepressor. Many of the resonances of the DNA shift on binding to the protein, which suggests changes in conformation throughout the sequence. The largest changes in shifts for the aromatic protons in the major groove are for A15 and G16, which are thought to hydrogen bond to the protein, possibly via water molecules. We have also examined the effect of DNA binding on the corepressor, tryptophan, in this complex. The indole proton resonance of the tryptophan undergoes a downfield shift of 1.2 ppm upon binding of DNA. This large shift is consistent with hydrogen bonding of the tryptophan to the phosphate backbone of the trpR operator DNA, as in the crystal structure of the holoprotein with the trp operator.Keywords: Trp repressor; deuteration ; trp operator.The Escherichia coli Trp repressor is activated by tryptophan to bind to at least five operators in the E. coli genome, namely the trpEDCBA (trpO), aroH, aroL, trpR and mtr operators (reviewed in [I]) [ 2 ] . It thereby represses initiation of transcription of genes involved in tryptophan uptake and biosynthesis in response to intracellular levels of tryptophan. As one of the smallest proteins whose binding to a specific DNA sequence is allosterically controlled, the Trp repressor has been studied extensively by a variety of genetic and physical methods; however, the basis for its DNA selectivity remains controversial.The structures of the protein and several of its complexes have been determined by X-ray crystallography [3-71 and, to lower resolution, by 'H and heteronuclear NMR spectroscopy [8-131. The protein belongs to the helix-turn-helix family of dimeric DNA-binding proteins. Tryptophan binds between the core of the molecule and the DNA-binding helices in each subunit, changing their orientation so that they are better positioned to bind to the DNA. In the crystal structure of the holoprotein bound to a double-stranded oligodeoxynucleotide containing a symmetrised version of the trpO operator sequence, the corepressor tryptophan also interacts directly with the phosphate backbone of the DNA [6]. Surprisingly in this structure there are no direct hydrogen bonds or van der Waals' interactions between the protein and the base pairs of the DNA known to be important for sequence-specific binding. There are several water-mediated interactions that could be important for specificity. Alternatively, specificity could arise from the interactions of the protein with the phosphate backbone ('indirect readout'). This result has been controversial: an NMR study of the same co...

Studies of the Escherichia coli Trp repressor binding to its five operators and to variant operator sequences

“…2). We studied protein binding to this sequence by 1 H‐NMR spectroscopy [20]. EMSA experiments carried out with DNA containing this sequence show retardation of DNA at similar concentrations of Trp repressor to those retarding trpR , but only one band of lower mobility than the free DNA is observed (Fig.…”

“…The tryptophan corepressor binds between the interlocked core of the dimer and the helix–turn–helix motif in each subunit, altering their orientation so as to improve DNA binding [16,17]. It also hydrogen bonds to the DNA backbone [18–20].…”

Binding of p53-derived Ligands to MDM2 Induces a Variety of Long Range Conformational Changes

“…In addition, chemical shifts can also be used to identify subtle conformational changes, situated away from the actual binding site, that the protein undergoes to accommodate the binding partner. 13 Here, we used NMR spectroscopy to investigate the structural changes in MDM2 on binding to different p53-derived peptides. We observed chemical shift changes throughout the whole protein domain, which are large enough to indicate changes in its overall conformation.…”

Solution Structure of the Aminoacyl-Capped Oligodeoxyribonucleotide Duplex (W-TGCGCAC)2

“…For partial intercalation, wedges built from combinations of aromatic and nonaromatic amino acids ( , ) or nonaromatic amino acids alone () are more typical. Examples for interactions of tryptophan side chains with DNA also include stacking on exposed nucleobases and hydrogen bonding with nucleobases or phosphodiesters ( − ), making it difficult to predict the DNA interactions of a tryptophan residue unconstrained by a given protein fold. For other indole derivatives, such as diaminidino-2-phenylindole (DAPI) (), the situation is similarly complex, since both intercalation and minor groove binding have been reported ( , ).…”