Abstract
The crystal structure of the complex of the BtuB receptor and the 135-residue coiled-coil receptor-binding R-domain of colicin E3 (E3R135) suggested a novel mechanism for import of colicin proteins across the outer membrane. It was proposed that one function of the R-domain, which extends along the outer membrane surface, is to recruit an additional outer membrane protein(s) to form a translocon for passage colicin activity domain. A 3.5-A crystal structure of the complex of E2R135 and BtuB (E2R135-BtuB) was obtained, which revealed E2R135 bound to BtuB in an oblique orientation identical to that previously found for E3R135. The only significant difference between the two structures was that the bound coiled-coil R-domain of colicin E2, compared with that of colicin E3, was extended by two and five residues at the N and C termini, respectively. There was no detectable displacement of the BtuB plug domain in either structure, implying that colicin is not imported through the outer membrane by BtuB alone. It was concluded that the oblique orientation of the R-domain of the nuclease E colicins has a function in the recruitment of another member(s) of an outer membrane translocon. Screening of porin knock-out mutants showed that either OmpF or OmpC can function in such a translocon. Arg(452) at the R/C-domain interface in colicin E2 was found have an essential role at a putative site of protease cleavage, which would liberate the C-terminal activity domain for passage through the outer membrane translocon.
Highlights
Protein transport across membranes in organelles and bacteria is known to involve multiprotein complexes [1, 2]
BtuB Is a Receptor but Not a Translocator for the Nuclease E Colicins—BtuB serves as the high affinity receptor that transfers the colicin from three-dimensional to the two-dimensional space of the outer membrane surface
It does not provide the pore, nor does it function alone to translocate the colicin across the outer membrane. This is inferred from (i) structure and (ii) electrophysiological data. (i) Displacement of the plug domain that would be necessary for entry of the colicin through the central pore was not seen in either the E2R135BtuB or the E3R135-BtuB structures
Summary
The crystal structure of the complex of the BtuB receptor and the 135-residue coiled-coil receptor-binding R-domain of colicin E3 (E3R135) suggested a novel mechanism for import of colicin proteins across the outer membrane. Colicin Ia [19], is associated with a “fishing rod” mechanism [16] Purification of BtuB—BtuB was expressed in strain TNE012 for recruitment of an outer membrane protein in addition to (pJC3) and purified using a previously published protocol [31]. The additional protein utilized to BtuB was extracted from the outer membrane with 1.5% (w/v) form a translocon for the import of nuclease E colicins is -D-octyl-glucoside, 50 mM Tris-HCl, 5 mM EDTA, phenylinferred to be the OmpF porin in most studies on colicins E2, methylsulfonyl fluoride/tosylphenylalanyl chloromethyl ketone.
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