Receptor-mediated import of nuclease E colicins

Protein import across cell membranes is an essential life process. Colicins are proteins produced and secreted by E. coli, which kill sibling cells that contain the translocation apparatus necessary for colicin import but do not produce an immunity protein to protect against that colicin. Colicins and toxins have a domain structure. Colicin E3, like all colicins, consists of three domains, the T-, R-, and C-domain that function in translocation, binding to the BtuB receptor, and cytotoxicity. The colicin is thought to enter the cell by utilizing a BtuB/OmpF translocon and requires an intracellular Tol system, including TolB, for cytotoxicity. The oblique orientation of the R-domain of E3 bound to BtuB seen in the E3R135·BtuB crystal structure, suggests that the initial binding to BtuB sequesters the colicin to the cell surface and uses the elongate R-domain to \u27fish\u27 for a second receptor-translocator OmpF. Subsequent events are: (i) binding to OmpF, and (ii) subsequent insertion to interact with TolB, (iii) unfolding of the T- and R-domains and release of immunity protein, (iv) proteolysis that releases the C- from the R-domain, and (v) entry of C-domain into the cytoplasm where it exerts its cytotoxicity. In the present studies, the mechanism of import of colicin was further refined and expanded to include other nuclease E colicins using a range of molecular biology, biochemical, electrophysiological and structural techniques. ^ The following conclusions were reached from these studies: (a) the structure of colicin E2R135·BtuB complex obtained at a resolution of 3.5 Å revealed the elongate R-domain to be bound at an angle to BtuB, as was found in the previously obtained 2.75 Å structure of E3R135·BtuB. (b) The residues involved in occlusion of OmpF channels were identified to be Asp5 and Arg7. (c) Both OmpF and OmpC can provide the translocation pore for nuclease E colicins. (d) The length of the N-terminal flexible peptide segment from residue 47-83 which allows E3 to interact with TolB while it is still bound to BtuB, was found to be important for cytotoxicity. (e) The proteolytic site and the nature of protease in the case of the RNase colicins was found be different from the DNase colicins. (f) About one-third of the single-gene knockout library of the E. coli genome has been screened for colicin cytotoxicity and a total of 364 new genes have been identified. The effect of colicins on the growth of single-gene knockout strains missing the kdpF, kdpA, surA or the citT gene is described. ^ These studies provided new insights into the import of colicins, especially nuclease E colicins. The new genomic information may lead to a new perspective on the mechanism of cellular import of the colicin proteins.