Channel Domain of Colicin A Modifies the Dimeric Organization of Its Immunity Protein

Abstract

Proteins conferring immunity against pore-forming colicins are localized in the Escherichia coli inner membrane. Their protective effects are mediated by direct interaction with the C-terminal domain of their cognate colicins. Cai, the immunity protein protecting E. coli against colicin A, contains four cysteine residues. We report cysteine cross-linking experiments showing that Cai forms homodimers. Cai contains four transmembrane segments (TMSs), and dimerization occurs via the third TMS. Furthermore, we observe the formation of intramolecular disulfide bonds that connect TMS2 with either TMS1 or TMS3. Co-expression of Cai with its target, the colicin A pore-forming domain (pfColA), in the inner membrane prevents the formation of intermolecular and intramolecular disulfide bonds, indicating that pfColA interacts with the dimer of Cai and modifies its conformation. Finally, we show that when Cai is locked by disulfide bonds, it is no longer able to protect cells against exogenous added colicin A.Pore-forming colicins are plasmid-encoded bacteriocins, synthesized by Enterobacteriacae, that are lethal to other related strains. Like many toxins, colicins are organized into structural domains that perform different functions: the Nterminal and the central domains are involved in the transport through the Escherichia coli envelope, and the C-terminal domain carries the toxic activity of the protein (1).The three-dimensional structures of the soluble form of the pore-forming domains of colicins A, E1, Ia, B, and N share the same general architecture: a bundle of eight amphipathic ␣-helices surrounding two hydrophobic ␣-helices (H8ϩH9), completely buried within the protein (2-6). However, crystal structures do not reveal the structure of the channel in the membrane, which remains uncharacterized. The hydrophobic helical hairpin may be the primary attachment region for channel formation and channel opening and closing require insertion and extrusion of an amphipathic segment, which has not been precisely defined (7,8).Each colicin plasmid also encodes a specific immunity protein that protects the producing cell against the cytotoxic activity of its colicin. Immunity proteins are integral inner membrane proteins. They are classified into two groups according to sequence similarities: the A type (immunity proteins to colicins A, B, N, and U) and the E1 type (immunity proteins to colicins E1, 5, K, 10, Ia, and Ib) (9 -11). The colicin A immunity protein (Cai) 2 has four transmembrane segments (TMSs), and its N and C termini are in the cytoplasm (Fig. 1), whereas the immunity protein to colicin E1 (Cei) crosses the cytoplasmic membrane three times, with the N terminus located in the cytoplasm and the C terminus in the periplasm (10, 11).The immunity proteins to pore-forming colicins diffuse laterally in the membrane and interact with helices of the pore-forming domain of their cognate colicin just prior channel opening (12-15). Genetic studies of A-type colicins indicate that the main determinant recognized by the immuni...

The Crystal Structure of the Lipid II-degrading Bacteriocin Syringacin M Suggests Unexpected Evolutionary Relationships between Colicin M-like Bacteriocins

“…In the case of the pore-forming and lipid II-degrading bacteriocins, complex formation with the immunity protein has not been demonstrated. These proteins are localized at the cytoplasmic membrane, where they negate the lethal effects of the toxin by mechanisms that are yet to be clearly delineated (14,15). In general, full protection is afforded only by the cognate immunity protein.…”

The crystal structure of the dimeric colicin M immunity protein displays a 3D domain swap

“…A recently published example for such an induced domain swapping event monitored in vivo is the colicin A immunity protein (Cai): cysteine crosslinking experiments revealed that the immunity protein (Cai) of the pore-forming colicin A which forms a homodimer in the membrane (Zhang et al, 2010). Cai contains four cysteines in the Table 2 Activity assay of Cmi constructs and mutants.…”

The ColM Family, Polymorphic Toxins Breaching the Bacterial Cell Wall

“…Instead, immunity to bacteriocins of the ColMα clade is provided by a different type of protein, PmiA, adopting a four-transmembrane-helix (TMH) topology ( Fig. 2A ) ( 24 ), an architecture that one would associate rather with immunity proteins for pore-forming bacteriocins ( 6 , 8 , 76 ). PmiAs are encoded downstream of the ColM-type pseudocin genes and essentially do not show sequence conservation, except for a short semiconserved motif that is predicted to be exposed to the periplasm.…”