Internal Dynamics of the Tryptophan Repressor (TrpR) and Two Functionally Distinct TrpR Variants, L75F-TrpR and A77V-TrpR, in Their l-Trp-Bound Forms

Abstract

Backbone amide dynamics of the Escherichia coli tryptophan repressor protein (WT-TrpR) and two functionally distinct variants, L75F-TrpR and A77V-TrpR, in their holo-(L-tryptophan (L-Trp) co-repressor-bound) form have been characterized using 15N NMR relaxation. The three proteins possess very similar structures ruling out major conformational differences as the source of their functional differences, and suggest that changes in protein flexibility are at the origin of their distinct functional properties. Comparison of site specific 15N-T1, 15N-T2, 15N-{1H}-nOes, reduced spectral density, and generalized order parameters (S2), indicates that backbone dynamics in the three holo-repressors are overall very similar with a few notable and significant exceptions for backbone atoms residing within the proteins’ DNA-binding domain. We find that flexibility is highly restricted for amides in core α-helices (i.e. helices A, B, C, and F), and a comparable “stiffening” is observed for residues in the DNA recognition helix (helix E) of the helix-D-turn-helix-E (HTH) DNA-binding domain of the three holo-repressors. Unexpectedly, amides located in helix D and in adjacent turn regions remain flexible. These data support the concept that residual flexibility in TrpR is essential for repressor function, DNA-binding, and molecular recognition of target operators. Comparison of the 15N NMR relaxation parameters of the holo-TrpRs with those of the apo-TrpRs indicate that the single point amino acid substitutions, L75F and A77V, perturb the flexibility of backbone amides of TrpR in very different ways, and are most pronounced in the apo-forms of the three repressors. Finally, we present these findings in the context of other DNA-binding proteins and the role of protein flexibility in molecular recognition.

Structural Studies of E73 from a Hyperthermophilic Archaeal Virus Identify the “RH3” Domain, an Elaborated Ribbon–Helix–Helix Motif Involved in DNA Recognition

“…Backbone amide 15 N NMR relaxation experiments including measurements of 15 N-T 1 , 15 N-T 2 , and heteronuclear 15 N-[ 1 H]-nOe were conducted in duplicate on a Bruker DRX 600 MHz solution NMR spectrometer using standard NMR pulse sequences (70–72) at 312 K and a pH of 5.0, in a manner analogous to our recent work with tryptophan repressor variants (73, 74). Briefly, 15 N-T 1 relaxation profiles were sampled at eight different relaxation delay time points of 40 ms, 96 ms, 200 ms, 400 ms, 600 ms, 1,000 ms, and 1,200 ms. 15 N-T 2 relaxation profiles were sampled at eight different relaxation delay periods of 8 ms, 16 ms, 32 ms, 40 ms, 64 ms, 80 ms, 104 ms, and 152 ms, with the delay between the series of 15 N 180 degree pulses applied in the CPMG sequence set to 0.5 ms (75, 76).…”

“…The resulting 15 N-T 1 , 15 N-T 2 , and 15 N-[ 1 H]-nOe data were then analyzed in terms of spectral density functions as described by Bracken et al (79) and more recently in Tripet et al (74). The data were also analyzed using the FastModelFree program (80) which interfaces with the ModelFree 4.0.1 program of Palmer and colleagues (81) and is based on the extended model-free formalism developed by Lipari and Szabo (82, 83).…”

“…All relaxation data were processed using NMRPipe, NMRDraw, and Sparky. 77,78 The resulting 15 N T 1 , 15 N T 2 , and 15 N− 1 H NOE data were then analyzed in terms of spectral density functions as described by Bracken et al 79 and more recently by Tripet et al 74 The data were also analyzed using FastModelFree, 80 °C with increasing amounts of E73 (from 0 to 20 μM). Loading buffer (5 μL of 20 mM Tris-HCl, 10 mM EDTA, and 50% glycerol) was added, and 25 μL of the sample mixture was analyzed on a 1.2% agarose gel with 0.5× TBE running buffer.…”

1H, 13C, 15N backbone and side chain NMR resonance assignments of the N-terminal NEAr iron transporter domain 1 (NEAT 1) of the hemoglobin receptor IsdB of Staphylococcus aureus

“…All NMR spectra were acquired at 298K (25°C) on a four-channel Bruker DRX 600 spectrometer with an inverse detection triple resonance ( 15 N, 13 C, 1 H) conventional NMR probe equipped with triple axis gradients, as previously described for other proteins of interest (Schlenker et al 2012; Tripet et al 2011). All data were processed and analyzed using NMRPipe Spectral Processing and Analysis System (Delaglio et al 1995) and Sparky NMR Assignment and Integration Software (Goddard and Kneller 2008).…”

Metabolic Engineering and Fermentation Process Strategies for L-Tryptophan Production by Escherichia coli

“…Researches have pointed out that inactivating TyrR resulted in a remarkable increase of DAHP synthase activity [68]. It is well-known that TrpR, a repressor encoded by a trpR gene located upstream of the tryptophan operator, regulates the transcription and translation of the tryptophan operator [69]. When the content of L-tryptophan in cells reaches a particular value, the transcription of the operator is stopped due to the inhibition of the repressor TrpR.…”