title: A22 disrupts the bacterial actin cytoskeleton by directly binding and inducing a low-affinity state in MreB.
abstract: S-(3,4-Dichlorobenzyl)isothiourea (A22) disrupts the actin cytoskeleton of bacteria, causing defects of morphology and chromosome segregation. Previous studies have suggested that the actin homologue MreB itself is the target of A22, but there has been no direct observation of A22 binding to MreB and no mechanistic explanation of its mode of action. We show that A22 binds MreB with at least micromolar affinity in its nucleotide-binding pocket in a manner that is sterically incompatible with simultaneous ATP binding. A22 negatively affects both the time course and extent of MreB polymerization in vitro in the presence of ATP. A22 prevents assembly of MreB into long, rigid polymers, as determined by both fluorescence microscopy and sedimentation assays. A22 increases the critical concentration of ATP-bound MreB assembly from 500 nM to approximately 2000 nM. We therefore conclude that A22 is a competitive inhibitor of ATP binding to MreB. A22-bound MreB is capable of polymerization, but with assembly properties that more closely resemble those of the ADP-bound state. Because the cellular concentration of MreB is in the low micromolar range, this mechanism explains the ability of A22 to largely disassemble the actin cytoskeleton in bacterial cells. It also represents a novel mode of action for a cytoskeletal drug and the first biochemical characterization of the interaction between a small molecule inhibitor of the bacterial cytoskeleton and its target.
authors.affiliation: Laboratory of Molecular Biology, University of Wisconsin, 1525 Linden Drive, Madison, Wisconsin 53706, USA.
authors.forename: G J
authors.initials: GJ
authors.lastname: Bean
authors.forename: S T
authors.initials: ST
authors.lastname: Flickinger
authors.forename: W M
authors.initials: WM
authors.lastname: Westler
authors.forename: M E
authors.initials: ME
authors.lastname: McCully
authors.forename: D
authors.initials: D
authors.lastname: Sept
authors.forename: D B
authors.initials: DB
authors.lastname: Weibel
authors.forename: K J
authors.initials: KJ
authors.lastname: Amann
mesh_terms: D000199:Actins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D003599:Cytoskeleton / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D011108:Polymers / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020124:Thermotoga maritima / Q000187:drug effects* / Q000378:metabolism; D013890:Thiourea / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; C435905:MreB1 protein, Thermotoga maritima; D011108:Polymers; D011994:Recombinant Proteins; C015799:2,6-dichlorobenzylthiopseudourea; D013890:Thiourea