Single-molecule force spectroscopy to decipher the early signalling step in membrane-bound penicillin receptors embedded into a lipid bilayer

Mescola, Andrea; Dauvin, Marjorie; Amoroso, Ana Maria; Duwez, Anne‐Sophie; Joris, Bernard

doi:10.1039/c9nr02466b

Cited by 5 publications

(7 citation statements)

References 41 publications

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“…The sensor domain lies right above the reentrant helix at the core of the gluzincin domain. This interaction is reinforced through binding of the L2 helix of the metalloprotease domain to the sensor domain, and is in agreement with single-molecule force spectroscopy assays that indicate a strong interaction between the sensor domain and the metalloprotease domain of the homologous protein BlaR1 from B. licheniformis 50 . Two observations lead us to propose that the model shown in Fig.…”

Section: Discussionsupporting

confidence: 84%

“…The connection between the β-strands of the sensor domain and the I 226 FWFNP turn provides a clear route for transmission of perturbed dynamics, through the reentrant helix directly to the metal site. Based on the observation that serine acylation at the sensor domain of BlaR1 from B. licheniformis detaches it from the L2 loop 50,53 , and our results that indicate a reduction in the susceptibility to proteolysis of the C-terminal end of the L2 loop in the presence of β-lactam antibiotics, we propose possible routes for subsequent activation of proteolytic action. When the sensor domain detaches from L2 due to steric overlap with the acylated β-lactam (orange spheres in Fig.…”

Section: Discussionmentioning

confidence: 60%

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An experiment-informed signal transduction model for the role of the Staphylococcus aureus MecR1 protein in β-lactam resistance

Belluzo

Abriata

Giannini

et al. 2019

Sci Rep

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The treatment of hospital- and community-associated infections by methicillin-resistant Staphylococcus aureus (MRSA) is a perpetual challenge. This Gram-positive bacterium is resistant specifically to β-lactam antibiotics, and generally to many other antibacterial agents. Its resistance mechanisms to β-lactam antibiotics are activated only when the bacterium encounters a β-lactam. This activation is regulated by the transmembrane sensor/signal transducer proteins BlaR1 and MecR1. Neither the transmembrane/metalloprotease domain, nor the complete MecR1 and BlaR1 proteins, are isolatable for mechanistic study. Here we propose a model for full-length MecR1 based on homology modeling, residue coevolution data, a new extensive experimental mapping of transmembrane topology, partial structures, molecular simulations, and available NMR data. Our model defines the metalloprotease domain as a hydrophilic transmembrane chamber effectively sealed by the apo-sensor domain. It proposes that the amphipathic helices inserted into the gluzincin domain constitute the route for transmission of the β-lactam-binding event in the extracellular sensor domain, to the intracellular and membrane-embedded zinc-containing active site. From here, we discuss possible routes for subsequent activation of proteolytic action. This study provides the first coherent model of the structure of MecR1, opening routes for future functional investigations on how β-lactam binding culminates in the proteolytic degradation of MecI.

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Section: Discussionsupporting

confidence: 84%

Section: Discussionmentioning

confidence: 60%

An experiment-informed signal transduction model for the role of the Staphylococcus aureus MecR1 protein in β-lactam resistance

Belluzo

Abriata

Giannini

et al. 2019

Sci Rep

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“…Notably, NMR study documented an association between the L2 loop peptide (as an amphiphilic helical segment) and the BlaR S protein . How the protease domain is activated is not understood, but the process must involve amplification of the conformational change that has crossed the membrane span . Loss of the BlaI repressor by proteolytic degradation of the now‐activated protease domain coincides with β‐lactamase expression in both B. licheniformis and S. aureus .…”

Section: Sensing and Evading The β‐Lactam Antibiotics By A Gram‐positmentioning

confidence: 99%

“…180 How the protease domain is activated is not understood, but the process must involve amplification of the conformational change that has crossed the membrane span. 181,182 Loss of the BlaI repressor by proteolytic degradation of the now-activated protease domain coincides with β-lactamase expression in both B. licheniformis 183,184 and S. aureus. [185][186][187] The protease domain activation is simultaneous to a pair of autoproteolytic reactions within the domain.…”

Section: Sensing and Evading The β-Lactam Antibiotics By A Gram-posmentioning

confidence: 99%

Constructing and deconstructing the bacterial cell wall

Fisher

Mobashery

2019

Protein Science

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The history of modern medicine cannot be written apart from the history of the antibiotics. Antibiotics are cytotoxic secondary metabolites that are isolated from Nature. The antibacterial antibiotics disproportionately target bacterial protein structure that is distinct from eukaryotic protein structure, notably within the ribosome and within the pathways for bacterial cell‐wall biosynthesis (for which there is not a eukaryotic counterpart). This review focuses on a pre‐eminent class of antibiotics—the β‐lactams, exemplified by the penicillins and cephalosporins—from the perspective of the evolving mechanisms for bacterial resistance. The mechanism of action of the β‐lactams is bacterial cell‐wall destruction. In the monoderm (single membrane, Gram‐positive staining) pathogen Staphylococcus aureus the dominant resistance mechanism is expression of a β‐lactam‐unreactive transpeptidase enzyme that functions in cell‐wall construction. In the diderm (dual membrane, Gram‐negative staining) pathogen Pseudomonas aeruginosa a dominant resistance mechanism (among several) is expression of a hydrolytic enzyme that destroys the critical β‐lactam ring of the antibiotic. The key sensing mechanism used by P. aeruginosa is monitoring the molecular difference between cell‐wall construction and cell‐wall deconstruction. In both bacteria, the resistance pathways are manifested only when the bacteria detect the presence of β‐lactams. This review summarizes how the β‐lactams are sensed and how the resistance mechanisms are manifested, with the expectation that preventing these processes will be critical to future chemotherapeutic control of multidrug resistant bacteria.

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“…The use of large colloidal or flat probes in indentation experiments allows the determination of the mechanical properties of a cell, mediating the result on a large contact area [12]. At the same time, the use of a sharp tip as indenter allows detection of local changes of mechanical properties [13,14] as well as investigation of single-molecule unfolding events [15]. The stiffer cytoskeletal filaments network [16] can be characterized by AFM, as well as the softer nuclear compartment [17].…”

Section: Introductionmentioning

confidence: 99%

Impact of Experimental Parameters on Cell–Cell Force Spectroscopy Signature

Oropesa‐Nuñez

Mescola

Vassalli

et al. 2021

Sensors

Self Cite

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Atomic force microscopy is an extremely versatile technique, featuring atomic-scale imaging resolution, and also offering the possibility to probe interaction forces down to few pN. Recently, this technique has been specialized to study the interaction between single living cells, one on the substrate, and a second being adhered on the cantilever. Cell–cell force spectroscopy offers a unique tool to investigate in fine detail intra-cellular interactions, and it holds great promise to elucidate elusive phenomena in physiology and pathology. Here we present a systematic study of the effect of the main measurement parameters on cell–cell curves, showing the importance of controlling the experimental conditions. Moreover, a simple theoretical interpretation is proposed, based on the number of contacts formed between the two interacting cells. The results show that single cell–cell force spectroscopy experiments carry a wealth of information that can be exploited to understand the inner dynamics of the interaction of living cells at the molecular level.

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Single-molecule force spectroscopy to decipher the early signalling step in membrane-bound penicillin receptors embedded into a lipid bilayer

Abstract: AFM-based single-molecule force spectroscopy is used to investigate the signalling mechanism of a penicillin receptor in a membrane environment.

Cited by 5 publications

References 41 publications

An experiment-informed signal transduction model for the role of the Staphylococcus aureus MecR1 protein in β-lactam resistance

An experiment-informed signal transduction model for the role of the Staphylococcus aureus MecR1 protein in β-lactam resistance

Constructing and deconstructing the bacterial cell wall

Impact of Experimental Parameters on Cell–Cell Force Spectroscopy Signature

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