EPR Spectroscopy Detects Various Active State Conformations of the Transcriptional Regulator CueR

Sameach, Hila; Ghosh, Shreya; Gevorkyan‐Airapetov, Lada; Saxena, Sunil; Ruthstein, Sharon

doi:10.1002/ange.201810656

Cited by 17 publications

(28 citation statements)

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“…12 We have also applied electron paramagnetic resonance (EPR) distance measurements to follow conformational changes that CueR undergoes as a function of Cu(I) and DNA binding in solution. 17,18 We revealed major changes in the structure, specifically between the apo-CueR-DNA and holo-CueR-DNA (with Cu(I)) complexes. In the holo-CueR-DNA complex (i.e., the active state), we were able to detect at least two different conformations of CueR in presence of Cu(I) and DNA.…”

Section: Introductionmentioning

confidence: 95%

“…The crystallographic data report changes in the DNA binding domain of the protein (Figure 1) between the repressed‐ and active‐state of CueR 12 . We have also applied electron paramagnetic resonance (EPR) distance measurements to follow conformational changes that CueR undergoes as a function of Cu(I) and DNA binding in solution 17,18 . We revealed major changes in the structure, specifically between the apo‐CueR‐DNA and holo‐CueR‐DNA (with Cu(I)) complexes.…”

Section: Introductionmentioning

confidence: 98%

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Allostery‐driven changes in dynamics regulate the activation of bacterial copper transcription factor

et al. 2022

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Metalloregulators bind and respond to metal ions by regulating the transcription of metal homeostasis genes. Copper efflux regulator (CueR) is a copper‐responsive metalloregulator that is found in numerous Gram‐negative bacteria. Upon Cu(I) coordination, CueR initiates transcription by bending the bound DNA promoter regions facilitating interaction with RNA polymerase. The structure of Escherichia coli CueR in presence of DNA and metal ion has been reported using X‐ray crystallography and cryo‐EM, providing information about the mechanism of action. However, the specific role of copper in controlling this transcription mechanism remains elusive. Herein, we use room temperature electron paramagnetic resonance (EPR) experiments to follow allosterically driven dynamical changes in E. coli CueR induced by Cu(I) binding. We suggest that more than one Cu(I) ion binds per CueR monomer, leading to changes in site‐specific dynamics at the Cu(I) binding domain and at the distant DNA binding site. Interestingly, Cu(I) binding leads to an increase in dynamics about 27 Å away at the DNA binding domain. These changes in the dynamics of the DNA binding domain are important for exact coordination with the DNA. Thus, Cu(I) binding is critical to initiate a series of conformational changes that regulate and initiate gene transcription. Broad audience statement The dynamics of metal transcription factors as a function of metal and DNA binding are complex. In this study, we use EPR spectroscopy to measure dynamical changes of Escherichia coli CueR as a function of copper and DNA binding. We show that copper controls the activation of the transcription processes by initiation a series of dynamical changes over the protein.

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Section: Introductionmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 98%

Allostery‐driven changes in dynamics regulate the activation of bacterial copper transcription factor

et al. 2022

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“…Cu II -nitrilotriacetic acid (Cu II –NTA) spin labeling of double histidine motifs for PD-EPR has been applied successfully to enzymes, 17 metalloproteins, 18 and nucleoprotein complexes. 19 …”

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Pulse Dipolar EPR Reveals Double-Histidine Motif Cu^II–NTA Spin-Labeling Robustness against Competitor Ions

Wort¹,

Arya

Ackermann³

et al. 2021

J. Phys. Chem. Lett.

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Pulse-dipolar EPR is an appealing strategy for structural characterization of complex systems in solution that complements other biophysical techniques. Significantly, the emergence of genetically encoded self-assembling spin labels exploiting exogenously introduced double-histidine motifs in conjunction with Cu II -chelates offers high precision distance determination in systems nonpermissive to thiol-directed spin labeling. However, the noncovalency of this interaction exposes potential vulnerabilities to competition from adventitious divalent metal ions, and pH sensitivity. Herein, a combination of room-temperature isothermal titration calorimetry (ITC) and cryogenic relaxation-induced dipolar modulation enhancement (RIDME) measurements are applied to the model protein Streptococcus sp. group G. protein G, B1 domain (GB1). Results demonstrate double-histidine motif spin labeling using Cu II -nitrilotriacetic acid (Cu II –NTA) is robust against the competitor ligand Zn II –NTA at >1000-fold molar excess, and high nM binding affinity is surprisingly retained under acidic and basic conditions even though room temperature affinity shows a stronger pH dependence. This indicates the strategy is well-suited for diverse biological applications, with the requirement of other metal ion cofactors or slightly acidic pH not necessarily being prohibitive.

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“…Cu 2+ is one of the most abundant cofactor metals in proteins, binding to several metalloproteins. Recently, Cu 2+ bound to two strategically placed histidine residues, also known as the Cu 2+ -dHis motif, have provided several significant advancements towards characterization of protein conformations [30][31][32][33]. The Cu 2+ -dHis motif provides nearly five-fold narrower distances than nitroxides, potentially enhancing the resolution of EPR distance methods [30,34].…”

Section: Introductionmentioning

confidence: 99%

Nanoscale spin detection of copper ions using double electron-electron resonance at room temperature

Zhang,

Ghosh,

Saxena

et al. 2021

Preprint

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We report the nanoscale spin detection and electron paramagnetic resonance (EPR) spectrum of copper ions via double electron-electron resonance with single spins in diamond at room temperature and low magnetic fields. We measure unexpectedly narrow EPR resonances with linewidths ∼ 2 − 3 MHz from small ensembles and single copper-chloride molecules dissolved in poly-lysine.We also observe coherent Rabi oscillations and hyperfine splitting from single Cu 2+ ions, which could be used for dynamic nuclear spin polarization and higher sensitivity of spin detection. We interpret and analyze these observations using both spin hamiltonian modeling of the copper-chloride molecules and numerical simulations of the predicted DEER response, and obtain a sensing volume ∼ (250nm) 3 . This work will open the door for copper-labeled EPR measurements under ambient conditions in bio-molecules and nano-materials.

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EPR Spectroscopy Detects Various Active State Conformations of the Transcriptional Regulator CueR

Abstract: This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record.

Cited by 17 publications

References 35 publications

Allostery‐driven changes in dynamics regulate the activation of bacterial copper transcription factor

Allostery‐driven changes in dynamics regulate the activation of bacterial copper transcription factor

Pulse Dipolar EPR Reveals Double-Histidine Motif Cu^II–NTA Spin-Labeling Robustness against Competitor Ions

Nanoscale spin detection of copper ions using double electron-electron resonance at room temperature

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EPR Spectroscopy Detects Various Active State Conformations of the Transcriptional Regulator CueR

Abstract: This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record.

Cited by 17 publications

References 35 publications

Allostery‐driven changes in dynamics regulate the activation of bacterial copper transcription factor

Allostery‐driven changes in dynamics regulate the activation of bacterial copper transcription factor

Pulse Dipolar EPR Reveals Double-Histidine Motif CuII–NTA Spin-Labeling Robustness against Competitor Ions

Nanoscale spin detection of copper ions using double electron-electron resonance at room temperature

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Pulse Dipolar EPR Reveals Double-Histidine Motif Cu^II–NTA Spin-Labeling Robustness against Competitor Ions