DNA-binding induced conformational change of c-Myb R2R3 analyzed using diffracted X-ray tracking

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Highlights

  • DNA-binding protein, c-Myb R2R3, largely fluctuates in solution.

  • We evaluated structural dynamics of R2R3 using diffracted X-ray tracking (DXT).

  • The flexibility of R2R3 is decreased upon the DNA binding.

  • We could determine the DNA-binding energies of R2R3 using DXT.

Abstract

Previous structural analyses have shown that R2R3, the minimum unit of the DNA-binding domain of the transcriptional factor c-Myb, is largely flexible in solution, and changes to a more rigid structure upon DNA binding. In this study, we evaluated the structural dynamics using the diffracted X-ray tracking method, in correlation with DNA-binding abilities under different salt conditions, and compared them with the previous results. The resultant curve of the mean square angular displacements (MSD) clearly showed that the flexibility of R2R3 was decreased upon DNA binding, and the DNA-binding energies determined using the angular diffusion coefficients were in good agreement with those determined using isothermal titration calorimetry. The results of the MSD curves also indicate that the translational length reduces by approximately half upon DNA binding.

Introduction

Structural fluctuations of a protein are closely correlated with its function. For example, when binding to another molecule, such as a ligand and a receptor, a protein changes its conformation or its conformational population shifts to the bound conformation [[1], [2], [3]]. These conformational changes or population shifts can be detected by structural analyses such as X-ray crystallography and NMR. While most methods could be used to determine static structures at high resolution, it is still difficult to determine structural dynamics on a temporal scale. Biophysical methods developed for the analysis of structural fluctuations have their own problems that need to be resolved, such as the unreliability of the data, requiring confirmation by other methods. Currently, single-molecule analysis is one of the promising methods for detecting the protein structure in solution. The sum of the protein structures determined by the single-molecule analysis should indicate the structural ensemble in solution. The diffracted X-ray tracking (DXT) can evaluate the protein structural fluctuations of the protein by detecting the movement of a gold-nanocrystal attached to the target protein [[4], [5], [6], [7], [8]]. Using a brilliant light source and high-speed detector, the structural dynamics of a protein can be detected in a time-dependent manner on the scale of μsec to msec. We applied the DXT method for the analysis of structural dynamics of a single-chain Fv antibody and a helix-bundle protein, and recently reported that the structural dynamics were changed by binding to another molecule, and the changes could be correlated with binding affinity as determined by isothermal titration calorimetry (ITC) experiments [7,8].

In this study, we analyzed the structural dynamics of the DNA-binding domain of c-Myb and the effects of DNA-binding using DXT. The c-myb protooncogene product, c-Myb, is a transcriptional activator that specifically binds to DNA fragments containing the consensus sequence PyAAC(G/T)G, where Py indicates a pyrimidine [9,10]. The DNA-binding domain of c-Myb consists of three imperfect 51- or 52-residue repeats (designated R1, R2, and R3 from the N terminus), and the last two repeats, R2 and R3, are sufficient for the recognition of the specific DNA sequences [11,12]. The solution structures of the specific DNA complex of R2R3, as well as R2R3 in its free form, were determined using NMR [[13], [14], [15]]. We have previously characterized the structural and functional properties of R2R3, using various biophysical methods such as NMR, surface plasmon resonance biosensor, ITC, and differential scanning calorimetry [[16], [17], [18], [19], [20], [21], [22], [23], [24], [25]]. One of the unique features of R2R3 is its structural flexibility under physiological conditions, which is critical for its specific DNA-binding function [17,22]. Overall, R2R3 fluctuates largely in solution, and becomes more rigid upon DNA binding. Here, we analyzed the structural dynamics of R2R3 in both DNA-free and -bound states by using DXT. In order to attach a gold-nanocrystal to the specific site of R2R3, a Met residue was introduced via a mutation into residue 135 located on the third helix of R2, and the residues Cys130 and Met189 were changed to Ile and Ala, respectively. Because this mutant C130I/H135M/M189A only has a sulfur-containing residue at 135, the time-dependent movement of a gold-nanocrystal attached to Met135 of R2R3 could be analyzed by DXT.

Section snippets

Materials

The DNA region encoding R2R3 on the plasmid, pRP23 [16], was first amplified as NdeI/EcoRI fragments by polymerase chain reaction (PCR), and the resultant PCR products were then cloned into pET28-a expression vector with an N-terminal polyhistidine-tag (His-tag). The plasmid of C130I/H135M/M189A R2R3 mutant was constructed by site-directed mutagenesis, and was overexpressed in Escherichia coli (E. coli) BL21 (DE3). Recombinant E. coli BL21 (DE3) harboring the expression vector was grown in

Results

The R2R3 mutant C130I/H135M/M189A was overexpressed in E. coli, and purified using a nickel-nitrilotriacetic acid matrix column. Purification yielded the protein with more than 95% purity, as estimated using SDS-PAGE analysis (data not shown). The far-UV CD spectra of the purified proteins in PBS and PBS with 250 mM NaCl indicated that they were properly folded and exhibited secondary structures, similar to that of the C130I standard protein reported previously [16,18,21] (Fig. 1).

DNA binding

Discussion

The minimum unit of the c-Myb DNA-binding domain R2R3 is largely flexible, with a fluctuating structure, like a ‘‘semi-intrinsically disordered’’ protein, which helps in finding a specific DNA binding site [27,28]. The previous structural and stability analyses have shown that R2 is more flexible and less stable than R3 [14,18,22]. R2 has an internal cavity in the hydrophobic core, which could be the main structural reason for its flexibility [14,17,29]. In this study, we designed a R2R3

Acknowledgements

The authors thank Ms Masako Hirose of Malvern Panalytical for technical support of ITC measurements. The CD measurements were performed in part at Osaka Prefecture University; MO is a visiting researcher. The synchrotron radiation experiments were performed at the BL40XU of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal No. 2016B1101 and 2017A1072). This work was supported by JSPS KAKENHI Grant Number 15J03567 to SI.

References (29)

Cited by (6)

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    The histograms of the angular displacement corresponding to θ, in the presence and absence of DNA, were analyzed for each 100 msec. In the absence of DNA, no differences were observed between the two immobilization densities (Fig. S1) [13]. In contrast, in the presence of DNA, two histogram peaks were observed at low immobilization density, whereas only one peak was observed at high immobilization density (Fig. 1).

  • Naïve balance between structural stability and DNA-binding ability of c-Myb R2R3 under physiological ionic conditions

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    The more fluctuating R2 is connected to the more rigid R3 via a linker which contributes to the cooperativity of R2 and R3 [6,23]. We observed that upon DNA-binding, the fluctuations decrease, resulting in an increased stability, as analyzed using differential scanning calorimetry, isothermal titration calorimetry (ITC), and diffracted X-ray tracking (DXT) experiments [20,23,24]. The DXT system could monitor the real-time motion by observations of X-ray diffracted spots from a gold-nanocrystal, tightly bound to the biomolecule [25,26].

1

These authors contributed equally to this work.

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