Tracking Disordered Extracellular Domains of Membrane Proteins in the Cell with Cu(II)-Based Spin Labels.

electron paramagnetic resonance (EPR) spectroscopy experiments provide high-resolution data about conformational changes of proteins within the cell. However, one of the limitations of EPR is the requisite of stable paramagnetic centers in a reducing environment. We recently showed that histidine-rich sites in proteins hold a high affinity to Cu(II) ions complexed with a chelator.

An efficient EPR spin-labeling method enables insights into conformational changes in DNA.

Electron paramagnetic resonance (EPR) is a powerful tool for elucidating both static and dynamic conformational alterations in macromolecules. However, to effectively utilize EPR for such investigations, the presence of paramagnetic centers, known as spin labels, is required. The process of spin labeling, particularly for nucleotides, typically demands intricate organic synthesis techniques.

The Dynamic Plasticity of P. aeruginosa CueR Copper Transcription Factor upon Cofactor and DNA Binding.

Bacteria use specialized proteins, like transcription factors, to rapidly control metal ion balance. CueR is a Gram-negative bacterial copper regulator. The structure of E.

Revealing the DNA Binding Modes of CsoR by EPR Spectroscopy.

In pathogens, a unique class of metalloregulator proteins, called gene regulatory proteins, sense specific metal ions that initiate gene transcription of proteins that export metal ions from the cell, thereby preventing toxicity and cell death. CsoR is a metalloregulator protein found in various bacterial systems that "sense" Cu(I) ions with high affinity. Upon copper binding, CsoR dissociates from the DNA promoter region, resulting in initiation of gene transcription.

An spin-labelling methodology provides structural information on cytoplasmic proteins in bacteria.

EPR in-cell spin-labeling was applied to CueR in . The methodology employed a Cu(II)-NTA complexed with dHis. High resolved in-cell distance distributions were obtained revealing minor differences between and in-cell data.

Conformations and Local Dynamics of the CopY Metal Sensor Revealed by EPR Spectroscopy.

Metal transcription factors regulate metal concentrations in eukaryotic and prokaryotic cells. Copper is a metal ion that is being tightly regulated, owing to its dual nature. Whereas copper is an essential nutrient for bacteria, it is also toxic at high concentrations.

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

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.

Dynamical interplay between the human high-affinity copper transporter hCtr1 and its cognate metal ion.

Abnormal cellular copper levels have been clearly implicated in genetic diseases, cancer, and neurodegeneration. Ctr1, a high-affinity copper transporter, is a homotrimeric integral membrane protein that provides the main route for cellular copper uptake. Together with a sophisticated copper transport system, Ctr1 regulates Cu(I) metabolism in eukaryotes.

Cellular Uptake of the ATSM-Cu(II) Complex under Hypoxic Conditions.

The Cu(II)-diacetyl-bis (N4-methylthiosemicarbazone) complex (ATSM-Cu(II)) has been suggested as a promising positron emission tomography (PET) agent for hypoxia imaging. Several in-vivo studies have shown its potential to detect hypoxic tumors. However, its uptake mechanism and its specificity to various cancer cell lines have been less studied.

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

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.

Cu(I) Controls Conformational States in Human Atox1 Metallochaperone: An EPR and Multiscale Simulation Study.

Atox1 is a human copper metallochaperone that is responsible for transferring copper ions from the main human copper transporter, hCtr1, to ATP7A/B in the Golgi apparatus. Atox1 interacts with the Ctr1 C-terminal domain as a dimer, although it transfers the copper ions to ATP7A/B in a monomeric form. The copper binding site in the Atox1 dimer involves Cys12 and Cys15, while Lys60 was also suggested to play a role in the copper binding.

Inhibiting the copper efflux system in microbes as a novel approach for developing antibiotics.

Five out of six people receive at least one antibiotic prescription per year. However, the ever-expanding use of antibiotics in medicine, agriculture, and food production has accelerated the evolution of antibiotic-resistant bacteria, which, in turn, made the development of novel antibiotics based on new molecular targets a priority in medicinal chemistry. One way of possibly combatting resistant bacterial infections is by inhibiting the copper transporters in prokaryotic cells.

Exploring the role of the various methionine residues in the Escherichia coli CusB adapter protein.

The dissemination of resistant pathogenic microbes has become one of the most challenging problems that modern medicine has faced. Developing novel drugs based on new molecular targets that previously were not targeted, is therefore the highest priority in antibiotics research. One approach that has been recently suggested is to inhibit copper transporters in prokaryotic systems.

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

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.

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

The interactions between proteins and their specific DNA sequences are the basis of many cellular processes. Hence, developing methods to build an atomic level picture of these interactions helps improve our understanding of key cellular mechanisms. CueR is an Escherichia coli copper-sensing transcription regulator.

The Neuronal Migration Factor srGAP2 Achieves Specificity in Ligand Binding through a Two-Component Molecular Mechanism.

srGAP proteins regulate cell migration and morphogenesis by shaping the structure and dynamics of the cytoskeleton and membranes. First discovered as intracellular effectors for the Robo1 axon-guidance receptor, srGAPs were later identified as interacting with several other nuclear and cytoplasmic proteins. In all these cases, the srGAP SH3 domain mediates protein-protein interactions by recognizing a short proline-rich segment on the cognate-binding partner.

Crystal structure of the extracellular juxtamembrane region of Robo1.

Robo receptors play pivotal roles in neurodevelopment, and their deregulation is implicated in several neuropathological conditions and cancers. To date, the mechanism of Robo activation and regulation remains obscure. Here we present the crystal structure of the juxtamembrane (JM) domains of human Robo1.

Role of Tim50 in the transfer of precursor proteins from the outer to the inner membrane of mitochondria.

Transport of essentially all matrix and a number of inner membrane proteins is governed, entirely or in part, by N-terminal presequences and requires a coordinated action of the translocases of outer and inner mitochondrial membranes (TOM and TIM23 complexes). Here, we have analyzed Tim50, a subunit of the TIM23 complex that is implicated in transfer of precursors from TOM to TIM23. Tim50 is recruited to the TIM23 complex via Tim23 in an interaction that is essentially independent of the rest of the translocase.

Interaction of Tim23 with Tim50 Is essential for protein translocation by the mitochondrial TIM23 complex.

The TIM23 complex is the major translocase of the mitochondrial inner membrane responsible for the import of essentially all matrix proteins and a number of inner membrane proteins. Tim23 and Tim50, two essential proteins of the complex, expose conserved domains into the intermembrane space that interact with each other. Here, we describe in vitro reconstitution of this interaction using recombinantly expressed and purified intermembrane space domains of Tim50 and Tim23.