A Model for Allosteric Communication in Drug Transport by the AcrAB-TolC Tripartite Efflux Pump.

RND family efflux pumps are complex macromolecular machines involved in multidrug resistance by extruding antibiotics from the cell. While structural studies and molecular dynamics simulations have provided insights into the architecture and conformational states of the pumps, the path followed by conformational changes from the inner membrane protein (IMP) to the periplasmic membrane fusion protein (MFP) and to the outer membrane protein (OMP) in tripartite efflux assemblies is not fully understood. Here, we investigated AcrAB-TolC efflux pump's allostery by comparing resting and transport states using difference distance matrices supplemented with evolutionary couplings data and buried surface area measurements. Our analysis indicated that substrate binding by the IMP triggers quaternary level conformational changes in the MFP, which induce OMP to switch from the closed state to the open state, accompanied by a considerable increase in the interface area between the MFP subunits and between the OMPs and MFPs. This suggests that the pump's transport-ready state is at a more favourable energy level than the resting state, but raises the puzzle of how the pump does not become stably trapped in a transport-intermediate state. We propose a model for pump allostery that includes a downhill energetic transition process from a proposed 'activated' transport state back to the resting pump

Data recording statistics and refinements (T = 100 K) for the OprN-wt and the OprN-Xe complex.

<p>Both are isomorphous, in the tetragonal I4 space group (Data from the highest resolution bin are in parentheses).</p

Views of the tripartite assembly of an efflux pump.

<p><b>A</b>: The schematic reconstitution complex supported by microscopy images on the MexAB-OprM in nanodiscs and by the chimeric AcrAB-TolC-AcrZ complex [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184045#pone.0184045.ref009" target="_blank">9</a>]. The oligomerization state is 3:6:3 for the three components, respectively. The RND part uses the proton motive force and is responsible of the active counter transport of the drug. <b>B</b>: The overall trimer assembly of OprN (the OMF component -in red at left- of the MexEF-OprN assembly). <b>C</b>: Topology of secondary structures of the OprN monomer with β-strands S1 to S4 and α-helices H1 to H7.</p

Stability, structural and functional properties of a monomeric, calcium-loaded adenylate cyclase toxin, CyaA, from Bordetella pertussis.

International audienceBordetella pertussis, the causative agent of whooping cough, secretes an adenylate cyclase toxin, CyaA, which invades eukaryotic cells and alters their physiology by cAMP overproduction. Calcium is an essential cofactor of CyaA, as it is the case for most members of the Repeat-in-ToXins (RTX) family. We show that the calcium-bound, monomeric form of CyaA, hCyaAm, conserves its permeabilization and haemolytic activities, even in a fully calcium-free environment. In contrast, hCyaAm requires sub-millimolar calcium in solution for cell invasion, indicating that free calcium in solution is involved in the CyaA toxin translocation process. We further report the first in solution structural characterization of hCyaAm, as deduced from SAXS, mass spectrometry and hydrodynamic studies. We show that hCyaAm adopts a compact and stable state that can transiently conserve its conformation even in a fully calcium-free environment. Our results therefore suggest that in hCyaAm, the C-terminal RTX-domain is stabilized in a high-affinity calcium-binding state by the N-terminal domains while, conversely, calcium binding to the C-terminal RTX-domain strongly stabilizes the N-terminal regions. Hence, the different regions of hCyaAm appear tightly connected, leading to stabilization effects between domains. The hysteretic behaviour of CyaA in response to calcium is likely shared by other RTX cytolysins

Author Correction: Structural and molecular determinants for the interaction of ExbB from Serratia marcescens and HasB, a TonB paralog.

International audienceAbstract ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The opportunistic pathogen Serratia marcescens ( Sm ) possesses both TonB and a heme-specific TonB paralog, HasB. ExbB Sm has a long periplasmic extension absent in other bacteria such as E. coli (Ec) . Long ExbB’s are found in several genera of Alphaproteobacteria, most often in correlation with a hasB gene. We investigated specificity determinants of ExbB Sm and HasB. We determined the cryo-EM structures of ExbB Sm and of the ExbB-ExbD Sm complex from S. marcescens . ExbB Sm alone is a stable pentamer, and its complex includes two ExbD monomers. We showed that ExbB Sm extension interacts with HasB and is involved in heme acquisition and we identified key residues in the membrane domain of ExbB Sm and ExbB Ec , essential for function and likely involved in the interaction with TonB/HasB. Our results shed light on the class of inner membrane energy machinery formed by ExbB, ExbD and HasB

Calcium-dependent disorder-to-order transitions are central to the secretion and folding of the CyaA toxin of Bordetella pertussis, the causative agent of whooping cough

International audienceThe adenylate cyclase toxin (CyaA) plays an essential role in the early stages of respiratory tract colonization by Bordetella pertussis, the causative agent of whooping cough. Once secreted, CyaA invades eukaryotic cells, leading to cell death. The cell intoxication process involves a unique mechanism of translocation of the CyaA catalytic domain directly across the plasma membrane of the target cell. Herein, we review our recent results describing how calcium is involved in several steps of this intoxication process. In conditions mimicking the low calcium environment of the crowded bacterial cytosol, we show that the C-terminal, calcium-binding Repeat-in-ToXin (RTX) domain of CyaA, RD, is an extended, intrinsically disordered polypeptide chain with a significant level of local, secondary structure elements, appropriately sized for transport through the narrow channel of the secretion system. Upon secretion, the high calcium concentration in the extracellular milieu induces the refolding of RD, which likely acts as a scaffold to favor the refolding of the upstream domains of the full-length protein. Due to the presence of hydrophobic regions, CyaA is prone to aggregate into multimeric forms in vitro, in the absence of a chaotropic agent. We have recently defined the experimental conditions required for CyaA folding, comprising both calcium binding and molecular confinement. These parameters are critical for CyaA folding into a stable, monomeric and functional form. The monomeric, calcium-loaded (holo) toxin exhibits efficient liposome permeabilization and hemolytic activities in vitro, even in a fully calcium-free environment. By contrast, the toxin requires sub-millimolar calcium concentrations in solution to translocate its catalytic domain across the plasma membrane, indicating that free calcium in solution is actively involved in the CyaA toxin translocation process. Overall, this data demonstrates the remarkable adaptation of bacterial RTX toxins to the diversity of calcium concentrations it is exposed to in the successive environments encountered in the course of the intoxication process