Exploring the interaction between the human copper transporter, CTR1, c-terminal domain and a methionine motif in the presence of Cu(I) and Ag(I) ions, using EPR spectroscopy

Shenberger, Yulia; Yarmiayev, Valeria; Ruthstein, Sharon

doi:10.1080/00268976.2013.807947

Cited by 10 publications

(21 citation statements)

References 45 publications

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“…EPR spectroscopy has emerged as an excellent tool for addressing the unclear aspects of Cu(I) transport cycle, since it does not require crystallization and does not depend on the protein size. EPR measurement can be performed in buffer solution, and even weak interactions between proteins can be detected [43]. EPR’s strength lies in its sensitivity to both atomic level changes and nanoscale fluctuations.…”

Section: Resultsmentioning

confidence: 99%

Unraveling the Impact of Cysteine-to-Serine Mutations on the Structural and Functional Properties of Cu(I)-Binding Proteins

Pavlin

Qasem

Sameach

et al. 2019

IJMS

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Appropriate maintenance of Cu(I) homeostasis is an essential requirement for proper cell function because its misregulation induces the onset of major human diseases and mortality. For this reason, several research efforts have been devoted to dissecting the inner working mechanism of Cu(I)-binding proteins and transporters. A commonly adopted strategy relies on mutations of cysteine residues, for which Cu(I) has an exquisite complementarity, to serines. Nevertheless, in spite of the similarity between these two amino acids, the structural and functional impact of serine mutations on Cu(I)-binding biomolecules remains unclear. Here, we applied various biochemical and biophysical methods, together with all-atom simulations, to investigate the effect of these mutations on the stability, structure, and aggregation propensity of Cu(I)-binding proteins, as well as their interaction with specific partner proteins. Among Cu(I)-binding biomolecules, we focused on the eukaryotic Atox1-ATP7B system, and the prokaryotic CueR metalloregulator. Our results reveal that proteins containing cysteine-to-serine mutations can still bind Cu(I) ions; however, this alters their stability and aggregation propensity. These results contribute to deciphering the critical biological principles underlying the regulatory mechanism of the in-cell Cu(I) concentration, and provide a basis for interpreting future studies that will take advantage of cysteine-to-serine mutations in Cu(I)-binding systems.

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Section: Resultsmentioning

confidence: 99%

Unraveling the Impact of Cysteine-to-Serine Mutations on the Structural and Functional Properties of Cu(I)-Binding Proteins

Pavlin

Qasem

Sameach

et al. 2019

IJMS

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“…18,25 EPR is much more sensitive than NMR, and can thus resolve even weak interaction between proteins, with only about 20% of the dissolved proteins in a complex. 47,48 In the presence of a metal ion, the amount of interacting proteins is higher, and can be resolved by less sensitive techniques. A close interaction between CusF and CusB was also observed in the chemical cross-linking experiments with and without Cu(I).…”

Section: Discussionmentioning

confidence: 99%

EPR spectroscopy identifies Met and Lys residues that are essential for the interaction between the CusB N-terminal domain and metallochaperone CusF

et al. 2015

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Copper plays a key role in all living organisms by serving as a cofactor for a large variety of proteins and enzymes involved in electron transfer, oxidase and oxygenase activities, and the detoxification of oxygen radicals. Due to its toxicity, a conserved homeostasis mechanism is required. In E. coli, the CusCFBA efflux system is a copper-regulating system and is responsible for transferring Cu(I) and Ag(I) out of the periplasm domain into the extracellular domain. Two of the components of this efflux system, the CusF metallochaperone and the N-terminal domain of CusB, have been thought to play significant roles in the function of this efflux system. Resolving the metal ion transport mechanism through this efflux system is vital for understanding metal- and multidrug-resistant microorganisms. This work explores one aspect of the E. coli resistance mechanism by observing the interaction between the N-terminal domain of CusB and the CusF protein, using electron paramagnetic resonance (EPR) spectroscopy, circular dichroism (CD), and chemical cross-linking. The data summarized here show that M36 and M38 of CusB are important residues for both the Cu(I) coordination to the CusB N-terminal domain and the interaction with CusF, and K32 is essential for the interaction with CusF. In contrast, the K29 residue is less consequential for the interaction with CusF, whereas M21 is mostly important for the proper interaction with CusF.

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“…EPR spectroscopy has emerged as an excellent tool for probing protein-protein interaction, since it does not require crystallization and does not depend on protein size. EPR can be measured in buffer solution, and even weak interactions between proteins can be detected [29]. EPR's strength lies in its sensitivity to both atomic level changes and nanoscale fluctuations.…”

Section: Probing the Interaction Between The Mbds Of Atp7b And The Mementioning

confidence: 99%

An EPR Study on the Interaction between the Cu(I) Metal Binding Domains of ATP7B and the Atox1 Metallochaperone

Zaccak

Qasem

Gevorkyan‐Airapetov

et al. 2020

IJMS

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Copper’s essentiality and toxicity mean it requires a sophisticated regulation system for its acquisition, cellular distribution and excretion, which until now has remained elusive. Herein, we applied continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) spectroscopy in solution to resolve the copper trafficking mechanism in humans, by considering the route travelled by Cu(I) from the metallochaperone Atox1 to the metal binding domains of ATP7B. Our study revealed that Cu(I) is most likely mediated by the binding of the Atox1 monomer to metal binding domain 1 (MBD1) and MBD4 of ATP7B in the final part of its extraction pathway, while the other MBDs mediate this interaction and participate in copper transfer between the various MBDs to the ATP7B membrane domain. This research also proposes that MBD1-3 and MBD4-6 act as two independent units.

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Exploring the interaction between the human copper transporter, CTR1, c-terminal domain and a methionine motif in the presence of Cu(I) and Ag(I) ions, using EPR spectroscopy

Cited by 10 publications

References 45 publications

Unraveling the Impact of Cysteine-to-Serine Mutations on the Structural and Functional Properties of Cu(I)-Binding Proteins

Unraveling the Impact of Cysteine-to-Serine Mutations on the Structural and Functional Properties of Cu(I)-Binding Proteins

EPR spectroscopy identifies Met and Lys residues that are essential for the interaction between the CusB N-terminal domain and metallochaperone CusF

An EPR Study on the Interaction between the Cu(I) Metal Binding Domains of ATP7B and the Atox1 Metallochaperone

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