A5057563515- Drug Transport and Resistance Mechanisms
- ATP Synthase and ATPases Research
- Mitochondrial Function and Pathology
- Bacterial Genetics and Biotechnology
- Antibiotic Resistance in Bacteria
- Enzyme Structure and Function
- RNA and protein synthesis mechanisms
- DNA and Nucleic Acid Chemistry
- Biochemical and Molecular Research
- Bacteriophages and microbial interactions
- HIV/AIDS drug development and treatment
- Protein Structure and Dynamics
- Trace Elements in Health
- Antibiotics Pharmacokinetics and Efficacy
- Mass Spectrometry Techniques and Applications
- Adenosine and Purinergic Signaling
- Hybrid Renewable Energy Systems
- Amino Acid Enzymes and Metabolism
- Advanced NMR Techniques and Applications
- Protein Interaction Studies and Fluorescence Analysis
- Lipid Membrane Structure and Behavior
- Cancer-related Molecular Pathways
- Peptidase Inhibition and Analysis
- Metalloenzymes and iron-sulfur proteins
- Biopolymer Synthesis and Applications
Centre National de la Recherche Scientifique
2016-2025
Université Claude Bernard Lyon 1
2016-2025
Institut de Biologie et de Chimie des Protéines
2002-2022
Laboratoire de Biochimie
2021-2022
Centre de Biologie Structurale
2022
Lyon College
2016-2022
Wenzhou Medical University
2018
Affiliated Hospital of Youjiang Medical University for Nationalities
2018
Chinese Academy of Medical Sciences & Peking Union Medical College
2018
Suzhou Institute of Systems Medicine
2018
A hexahistidine-tagged C-terminal nucleotide-binding domain (H 6 -NBD2) from mouse P-glycoprotein was designed, overexpressed, and purified as a highly soluble recombinant protein. Intrinsic fluorescence of its single tryptophan residue allowed monitoring high-affinity binding 2′(3′)- N -methylanthraniloyl-ATP (MANT-ATP), fluorescent ATP derivative that induces marked quenching correlated to resonance-energy transfer. H -NBD2 also bound all flavonoids known modulate the multidrug resistance...
Human cytomegalovirus (HCMV) infection stimulates cellular DNA synthesis and causes chromosomal damage. Because such events likely affect proliferation, we investigated the impact of HCMV on key components cell cycle. Early after infection, induced elevated levels cyclin E, E-associated kinase activity, two tumor suppressor proteins, p53 retinoblastoma gene product (Rb). The steady-state concentration Rb continued to rise throughout with most protein remaining in highly phosphorylated form....
Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved three ATP-Mg2+-bound outward-facing conformations Bacillus subtilis (homodimeric) BmrA x-ray crystallography and single-particle cryo-electron microscopy (EM) in detergent solution, one them with rhodamine 6G (R6G), substrate exported when overexpressed B. subtilis. Two R6G molecules bind to drug-binding cavity at level outer...
ATP-binding cassette (ABC) proteins constitute one of the widest families in all organisms, whose P-glycoprotein involved resistance cancer cells to chemotherapy is an archetype member. Although three-dimensional structures several nucleotide-binding domains ABC are now available, catalytic mechanism triggering functioning these still remains elusive. In particular, it has been postulated that ATP hydrolysis proceeds via acid-base catalyzed by Glu residue adjacent Walker-B motif (Geourjon,...
The involvement of transporters in multidrug resistance bacteria is an increasingly challenging problem, and most the pumps identified so far use protonmotive gradient as energy source. A new member ATP-binding cassette (ABC) family, known Bacillus subtilis YvcC homologous to each half mammalian P-glycoprotein LmrA Lactococcus lactis, has been studied here. yvcC gene was constitutively expressed B. throughout its growth, a knockout mutant showed lower rate ethidium efflux than wild-type...
The study of membrane proteins remains a challenging task, and approaches to unravel their dynamics are scarce. Here, we applied hydrogen/deuterium exchange (HDX) coupled mass spectrometry probe the motions bacterial multidrug ATP-binding cassette (ABC) transporter, BmrA, in inward-facing (resting state) outward-facing (ATP-bound) conformations. Trypsin digestion global or local HDX support transition between inward- conformations during catalytic cycle BmrA. However, resting state, peptides...
Background Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason fact that detergents do not stabilize membrane domains as efficiently natural lipids in membranes, often leading to a partial complete loss activity/stability during protein extraction and purification preventing crystallization an active conformation. Methodology/Principal Findings Anionic calix[4]arene based (C4Cn, n = 1–12) were...
Abstract Most membrane proteins studies require the use of detergents, but because lack a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional structural data analyses. To solve this problem, we propose based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) allows quantification pure or mixed detergents in complex with proteins. We validated wide variety automated process, thereby allowing routine for...
Carbon catabolite repression allows bacteria to rapidly alter the expression of catabolic genes in response availability metabolizable carbon sources. In Bacillus subtilis, this phenomenon is controlled by HPr kinase (HprK) that catalyzes ATP-dependent phosphorylation either (histidine containing protein) or Crh (catabolite HPr) on residue Ser-46. We report here B. subtilis HprK forms homo-oligomers constituted most likely eight subunits. Related complex structure, enzyme displays strong...
The bovine heart mitochondrial F1-ATPase depleted of nucleotides (nd-MF1) hydrolyzes 50 microM ATP in three kinetic phases at 30 degrees C. An initial burst rapidly transforms into an intermediate, slower rate, which slowly accelerates to the final, steady-state rate. intermediate phase disappears progressively as concentration assay medium is increased and absent 2 mM. Activation lost when nd-MF1 inactivated by 5'-p-fluorosulfonylbenzoyladenosine, modifies noncatalytic sites. Correlation...
All bacterial multidrug ABC transporters have been shown to work as either homodimers or heterodimers. Two possibly linked genes, patA and patB from Streptococcus pneumococcus, that encode half-ABC previously be involved in fluoroquinolone resistance. We showed the ΔpatA, ΔpatB, ΔpatA/ΔpatB mutant strains were more sensitive unstructurally related compounds, i.e., ethidium bromide fluoroquinolones, than wild-type R6 strain. Inside-out vesicles prepared Escherichia coli expressing PatA and/or...
Background The phylum Crenarchaeota lacks the FtsZ cell division hallmark of bacteria and employs instead Cdv proteins. While CdvB CdvC are homologues eukaryotic ESCRT-III Vps4 proteins, implicated in membrane fission processes during multivesicular body biogenesis, cytokinesis budding some enveloped viruses, little is known about structure function CdvA. Here, we report biochemical biophysical characterization three proteins from hyperthermophilic archaeon Metallospherae sedula....
Abstract Overexpression of correctly folded membrane proteins is a fundamental prerequisite for functional and structural studies. One the most commonly used expression systems production Escherichia coli . While misfolded typically aggregate form inclusions bodies, that are addressed to extractable by detergents generally assumed be properly folded. Accordingly, GFP fusion strategy often as fluorescent proxy monitor their folding quality. Here we investigated functionality two different...
The detailed mechanism of ATP hydrolysis in ATP-binding cassette (ABC) transporters is still not fully understood. Here, we employed 31P solid-state NMR to probe the conformational changes and dynamics during catalytic cycle by locking multidrug ABC transporter BmrA pre-hydrolytic, transition post-hydrolytic states, using a combination mutants analogues. spectra reveal that binds strongly pre-hydrolytic state both sites as inferred from analysis non-hydrolytic E504A mutant. In wild-type...
GTPases have been demonstrated to be necessary for the proper assembly of ribosome in bacteria and eukaryotes. Here, we show that essential YphC YsxC are required large ribosomal subunit biogenesis Bacillus subtilis. Sucrose density gradient centrifugation subunits isolated from YphC-depleted cells YsxC-depleted indicates they similar 45S intermediate previously identified RbgA-depleted cells. The sedimentation large-subunit was identical found cells, while sedimented slightly slower than...
ATP-binding cassette (ABC) transporters are involved in the transport of a wide variety substrates, and ATP-driven dimerization their nucleotide binding domains (NBDs) has been suggested to be one most energetic steps catalytic cycle. Taking advantage propensity BmrA, bacterial multidrug resistance ABC transporter, form stable, highly ordered ring-shaped structures [Chami et al. (2002) J. Mol. Biol. 315, 1075−1085], we show here that addition ATP presence Mg2+ prevented ring formation or...
Abstract ATP-binding-cassette (ABC) transporters are molecular pumps that translocate molecules across the cell membrane by switching between inward-facing and outward-facing states. To obtain a detailed understanding of their mechanism remains challenge to structural biology, as these proteins notoriously difficult study at level in active, membrane-inserted form. Here we use solid-state NMR investigate multidrug ABC transporter BmrA reconstituted lipids. We identify chemical-shift...
ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity substrates across biological membranes. Some them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite wealth structural data gained in last two decades, molecular mechanism these efflux pumps remains elusive, including extent separation between nucleotide-binding domains (NBDs) during transport cycle....
Bacterial genomics have revealed the widespread occurrence of eukaryotic-like protein kinases in prokaryotes, but little is known about their regulation, endogenous substrates, and physiological role. The present study concerns one these enzymes, serine/threonine kinase PknF from Mycobacterium tuberculosis. It shown that, addition to its autokinase activity, able phosphorylate Rv1747, a newly described ABC transporter. This reaction appears involve two FHA domains Rv1747. suggested that...