Flexibility in the receptor-binding domain of the enzymatic colicin E9 is required for toxicity against Escherichia coli cells

Abstract

The events that occur after the binding of the enzymatic E colicins to Escherichia coli BtuB receptors that lead to translocation of the cytotoxic domain into the periplasmic space and, ultimately, cell killing are poorly understood. It has been suggested that unfolding of the coiled-coil BtuB receptor binding domain of the E colicins may be an essential step that leads to the loss of immunity protein from the colicin and immunity protein complex and then triggers the events of translocation. We introduced pairs of cysteine mutations into the receptor binding domain of colicin E9 (ColE9) that resulted in the formation of a disulfide bond located near the middle or the top of the R domain. After dithiothreitol reduction, the ColE9 protein with the mutations L359C and F412C (ColE9 L359C-F412C) and the ColE9 protein with the mutations Y324C and L447C (ColE9 Y324C-L447C) were slightly less active than equivalent concentrations of ColE9. On oxidation with diamide, no significant biological activity was seen with the ColE9 L359C-F412C and the ColE9 Y324C-L447C mutant proteins; however diamide had no effect on the activity of ColE9. The presence of a disulfide bond was confirmed in both of the oxidized, mutant proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The loss of biological activity of the disulfide-containing mutant proteins was not due to an indirect effect on the properties of the translocation or DNase domains of the mutant colicins. The data are consistent with a requirement for the flexibility of the coiled-coil R domain after binding to BtuB.

FIG. 1.

Structure of ColE3 (35) with the location of the disulfide bond formed between the C324-C447 and C359-C412 mutations in the R domain of ColE9 indicated in spacefill.

FIG. 2.

Growth inhibitory activity of disulfide lock mutant proteins. The growth inhibitory activity of the ColE9 L359C-F412C, ColE9 Y324C-L447C, and ColE9 Y324C-V405C mutant proteins is shown compared to ColE9 in a large-plate assay. Aliquots of doubling dilutions of each of the proteins, containing concentrations of between 0.6 nM and 1.2 μM, were spotted onto a large agar plate spread with the indicator E. coli DH5α. A clear zone in the lawn of cells indicates growth inhibition by the colicin protein at that dilution.

FIG. 3.

Growth inhibitory activity in liquid medium. (A) The effect on E. coli DH5α cells grown in LB medium, with no additions (○), with 3 nM ColE9 (•), with 30 nM oxidized ColE9 Y324C-L447C mutant protein (□), with 30 nM reduced ColE9 Y324C-L447C mutant protein (▪), with 30 nM oxidized ColE9 L359C-F412C mutant protein (▵), or with 30 nM reduced ColE9 L359C-F412C mutant protein (▴). (B) The effect on E. coli DH5α cells grown in LB medium, with no additions (○), with 1 mM DTT (⋄), with 3 nM ColE9 (•), with 30 nM oxidized ColE9 Y324C-L447C mutant protein plus 1 mM DTT (▪), or with 30 nM oxidized ColE9 L359C-F412C mutant protein plus 1 mM DTT (▴). OD₆₀₀, optical density at 600 nm.

FIG. 4.

Presence of a disulfide confirmed by MALDI-TOF. The spectra of the oxidized (right) and reduced (left) ColE9 L359C-F412C mutant protein is shown. The sequence and residue numbers of a disulfide-containing peptide observed in the oxidized ColE9 L359C-F412C protein spectra are indicated at the top of the figure.

FIG. 5.

Assay of BtuB receptor binding. In vivo competition assay using E. coli DH5α cells incubated with no additions (○), with 3 nM ColE9 (•), with 300 nM ColE9 L359C-F412C mutant protein (▪), and with 3 nM ColE9 and the ColE9 L359C-F412C mutant protein at a ratio of 1:1 (▵), 1:10 (▴), or 1:100 (X). OD₆₀₀, optical density at 600 nm.

FIG. 6.

DNase activity assays. The DNase activities of the oxidized (▵) and reduced (▴) ColE9 L359C-F412C and the oxidized (□) and reduced (▪) ColE9 Y324C-L447C mutant proteins were compared to that of ColE9 (•) by using the Kunitz assay. The Im9 protein was removed from the ColE9 proteins to allow the DNase assay to be performed. OD₂₆₀, optical density at 260 nm.