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.

Colicin Biology

“…This implies structural modifications starting from the receptor binding domain (237,607). This suggestion correlates with the inactivity of oxidized forms of double-cysteine mutants that lock the colicin E9 reception domain without interfering with its capacity to interact with the BtuB receptor (513). Overall, these data suggest that the unfolding of the receptor binding domain is necessary for starting the translocation process.…”

“…Data are available concerning structural modifications of colicins or Tol subunits during the transit step, suggesting that it is not a passive process involving sequential interactions with cell envelope components but rather that it is a dynamic process involving conformational changes of both the machinery components and the ligand. First, it has been shown that the colicin A central domain remains in an interaction with the BtuB receptor while both N-and C-terminal domains are translocated (24, 165,513). This implies that the three domains are linked via flexible regions that are subject to large-scale structural modifications.…”

“…The notion that unfolding facilitates killing has been shown experimentally: colicin A was shown to kill cells more quickly when denatured with urea than the natively folded protein (24), and disulfide bonds engineered into the pore-forming domain delayed translocation (165). Similarly, a disulfide-bonded colicin E9 mutant, linked near the end of its coiled coil (close to the nuclease domain), was inactive unless a reductant was present (513). Colicin unfolding would presumably require energy, but this is likely to occur sometime after binding to the receptor.…”

Rapid Detection of Colicin E9-Induced DNA Damage Using Escherichia coli Cells Carrying SOS Promoter- lux Fusions

“…This protein contains two cysteine mutations in the ColE9 receptor-binding domain that, in the reduced protein, do not affect its colicin activity. Formation of a disulfide bond under oxidizing conditions leads to loss of biological activity, as previously described (22). Plasmid pAG1 is derived from pML261 and contains a 2.4-kb EcoRIHindIII fragment that encodes the complete btuB gene in the vector pUC8 (15).…”

“…(ii) Synchronized cell death using a "disulfide-locked" colicin E9 protein. We recently presented data showing that a mutant ColE9 protein (BH29) with an engineered disulfide bond in its receptor-binding domain (via two strategically positioned cysteine mutations) was inactive in the oxidized form and that activity was restored when it was reduced through the addition of DTT (22). The current reporter assay, because of its faster response time and sensitivity, enabled us to probe the early events after DTT addition.…”