ABSTRACT
The small protein AcrZ in Escherichia coli interacts with the transmembrane portion of the multidrug efflux pump AcrB and increases resistance of the bacterium to a subset of the antibiotic substrates of that transporter. It is not clear how the physical association of the two proteins selectively changes activity of the pump for defined substrates. Here, we report cryo-EM structures of AcrB and the AcrBZ complex in lipid environments, and comparisons suggest that conformational changes occur in the drug-binding pocket as a result of AcrZ binding. Simulations indicate that cardiolipin preferentially interacts with the AcrBZ complex, due to increased contact surface, and we observe that chloramphenicol sensitivity of bacteria lacking AcrZ is exacerbated when combined with cardiolipin deficiency. Taken together, the data suggest that AcrZ and lipid cooperate to allosterically modulate AcrB activity. This mode of regulation by a small protein and lipid may occur for other membrane proteins.
Subject(s)
Cardiolipins/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Multidrug Resistance-Associated Proteins/chemistry , Multidrug Resistance-Associated Proteins/metabolism , Allosteric Regulation , Binding Sites , Carrier Proteins/genetics , Chloramphenicol/pharmacology , Cryoelectron Microscopy , Crystallography, X-Ray , Drug Resistance, Bacterial , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Models, Molecular , Multiprotein Complexes/chemistry , Protein Binding , Protein Conformation , Substrate SpecificityABSTRACT
Positioning of the division site in many bacterial species relies on the MinCDE system, which prevents the cytokinetic Z-ring from assembling anywhere but the mid-cell, through an oscillatory diffusion-reaction mechanism. MinD dimers bind to membranes and, via their partner MinC, inhibit the polymerization of cell division protein FtsZ into the Z-ring. MinC and MinD form polymeric assemblies in solution and on cell membranes. Here, we report the high-resolution cryo-EM structure of the copolymeric filaments of Pseudomonas aeruginosa MinCD. The filaments consist of three protofilaments made of alternating MinC and MinD dimers. The MinCD protofilaments are almost completely straight and assemble as single protofilaments on lipid membranes, which we also visualized by cryo-EM.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Pseudomonas aeruginosa/metabolism , Cryoelectron Microscopy , Cytoskeletal Proteins/metabolism , Membrane Lipids/metabolism , Models, Molecular , Multiprotein Complexes/chemistry , Protein Conformation , Protein Multimerization , Pseudomonas aeruginosa/chemistryABSTRACT
Low copy-number plasmid pLS32 of Bacillus subtilis subsp. natto contains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB, and the centromeric sequence parN Similar to the ParMRC partitioning system from Escherichia coli plasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids through interactions with AlfB and parN Because, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of nonbundling AlfA filaments to 3.4-Å resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin fold has been accommodated by unique longitudinal and lateral contacts, while still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments, we obtained a pseudoatomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly antiphasic with near eightfold helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, how deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerization, nucleotide hydrolysis, and antiparallel doublet formation are retained to fulfill the system's biological raison d'être.
Subject(s)
Actin Cytoskeleton/ultrastructure , Bacillus subtilis/metabolism , Bacillus subtilis/ultrastructure , Bacterial Proteins/ultrastructure , Cryoelectron Microscopy/methods , Plasmids , Actin Cytoskeleton/metabolism , Bacterial Proteins/metabolism , Crystallography, X-Ray , Cytoskeleton/metabolism , DNA, Bacterial , Models, MolecularABSTRACT
Most bacteria and archaea contain filamentous proteins and filament systems that are collectively known as the bacterial cytoskeleton, though not all of them are cytoskeletal, affect cell shape, or maintain intracellular organization. To view this SnapShot, open or download the PDF.
