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.

Figure 1. A22-MreB interaction observed by NMR

5 mM A22 was incubated with varying concentrations of MreB in 10 mM Tris-HCl, pH 8.0 at 10°C. Normalized integrated peak areas collected for the three aromatic protons in A22 were plotted versus protein concentration and fitted to a one-site binding model. The Kd was found to be 1.32 ± 0.14 µM (R² = 0.89).

Figure 2. A22 docking in the MreB nucleotide cleft

(A) Docking site of A22 in the nucleotide-free form of MreB. (B) Details of the binding site of A22 in MreB with residues within 2.5 Å of A22 shown with their sidechains. ATP is shown in its crystal position, illustrating the clashes that would occur between its beta and gamma phosphates and A22.

Figure 3. A22-mediated inhibition of MreB assembly visualized by fluorescence microscopy

1 µM MreB (20% Alexa-488 labeled) was polymerized in 10 mM imidazole, pH 7.0, 1 mM MgCl₂, 1 mM EGTA, 20 mM KCl and 200 µM ATP at 20° C for 1 hr in the absence (A) or presence (B) of 300 µM A22 and imaged directly by epifluorescence microscopy. Scale bar = 10 µm.

Figure 4. Effects of A22 on MreB polymerization timecourse

5 µM MreB was polymerized in 10 mM imidazole, pH 7.0, 20 mM KCl, 1 mM MgCl₂ and 200 µM ATP at 20° C in the presence of varying concentrations of A22. MreB polymerization timecourse was followed by 400 nm right angle light scattering. A22 concentrations are as specified in the inset.

Figure 5. Effect of A22 on MreB critical concentration

Varying concentrations of MreB were polymerized at 4° C overnight in 10 mM imidazole, pH 7.0, 1 mM MgCl₂, 1 mM EGTA, 20 mM KCl and 200 µM ATP in the absence (A) or presence (B) of 300 µM A22. Samples were equilibrated to 20° C for 1 h and the 400 nm light scattering intensity was measured. Linear fits to the data yielded critical concentrations of 500 nM (A) and 2000 nM (B), respectively.

Figure 5. Effect of A22 on MreB critical concentration

Varying concentrations of MreB were polymerized at 4° C overnight in 10 mM imidazole, pH 7.0, 1 mM MgCl₂, 1 mM EGTA, 20 mM KCl and 200 µM ATP in the absence (A) or presence (B) of 300 µM A22. Samples were equilibrated to 20° C for 1 h and the 400 nm light scattering intensity was measured. Linear fits to the data yielded critical concentrations of 500 nM (A) and 2000 nM (B), respectively.

Figure 6. Effects of A22 on sedimentation of MreB

5 µM MreB was polymerized for 1 h at 4° C or 20° C in 10 mM imidazole, pH 7.0, 1 mM MgCl₂, 1 mM EGTA, 20 mM KCl and 200 µM ATP in the presence or absence of 100 µM A22. Samples were centrifuged at 4° C or 20° C for 30 min at 100000g. Equivalent volumes of total (T), supernatant (S) and pellet (P) samples are shown by SDS-PAGE and Coomassie blue.