Immunity proteins against Colicins (EcCIP) are produced by E. coli alongside the relevant colicin protein (EcCol) to protect the cell from the cytotoxic domain of the colicin. Usually this involves binding to and blocking the active site of the domain, to prevent it from targeting the cells own mechanisms.
Once released from the producing cell, the immunity proteins are no longer needed, as the cytotoxic domain needs to be active once a target cell has been penetrated. Often it is disassociated from the colicin upon binding to an outer membrane receptor on the target cell. Find more details in
Directed evolution and Colicin7/Immunity-proteins complexes[1]
Iterative rounds of random mutagenesis and selection of immunity protein 9 (colored yellow) toward higher affinity for ColE7, and selectivity (against ColE9 inhibition), led to significant increase in affinity and selectivity. Several evolved variants were obtained. The crystal structures of the two final generation R12-2 (3gkl; T20A, N24D, T27A, S28T, V34D, V37J, E41G, and K57E) and R12-13 (3gjn; N24D, D25E, T27A, S28T, V34D, V37J, and Y55W) in complex with ColE7 were solved.
of the immunity protein 9 (Im9, 1bxi, colored yellow), evolved variant R12-2 (lime), and immunity protein 7 (Im7, colored blue, 7cei) reveals their structural identity. However, when the immunity proteins-bound , they demonstrate somewhat different picture. The Im9 and Im7 are differ more in their binding configurations (19°, with Tyr54-Tyr55 as the pivot), while the variant R12-2 is in an intermediate configuration between Im9 and Im7. Of note, in the variant R12-2 (3gkl) and Im9 (1bxi) there are Tyr54 and Tyr55, while in the Im7 (7cei) Tyr55 and Tyr56 are homologous to them. The most are in the loop between helices α1 and α2 in Im9 (yellow, labeled in black) and evolved variant R12-2 (lime, labeled in black). This loop consists of three mutations: N24D, T27A, and S28T in variant R12-2. We can see the deviations in the relative position of helices α1 and α2, in the loop's backbone and in the side chains of residues 24, 26 and 28.
Comparison of the different Im-colicin complexes reveals changes in the binding configuration of the evolved variants which increase affinity toward ColE7 by re-aligning pre-existing Im9 residues. Glu30 of Im9 (1bxi, colored yellow) forms with Arg54 of ColE9 (orange), whereas Asp51 have not direct side chain–side chain interactions. Asp31 of Im7 (blue) which is corresponding to Im9 Glu30 is involved in to Arg520 and Lys525 of ColE7 (darkmagenta), while Asp52 of Im7 (corresponding to Im9 Asp51) is within hydrogen bond distance to Thr531 and Arg530 of ColE7 (7cei). Glu30 in the variant R12-2 (lime) is shifted and forms a to Arg520 of ColE7 (magenta). Asp51 is within hydrogen bond distance to Thr531 of ColE7 (3gkl). However, the side chains of Lys525 and Arg530, which are very important in salt bridge contacts with Glu30 and Asp51, respectively, in the structure of the ColE7–Im7 complex have a different conformation that eliminates these contacts in evolved variant R12-2.
In the Val37 (colored magenta) forms stabilizing hydrogen bond with Leu33. In the , Ile37 (colored darkmagenta) interacts with two additional residues, Tyr54 and Ser50. Moreover, Ile37 also forms additional hydrogen bond with Gly41 and can thereby have enabled the appearance of the selectivity mutation E41G.
In contrast to the evolved variant R12-2 (3gkl), the evolved variant R12-13 (3gjn) carries the in the conserved region. Both Tyr55 in R12-2 and Trp55 in R12-13 could sustain the
hydrophobic core and create a to Lys528 backbone (3gkl colicin residues are colored in magenta, 3gjn colicin residues are colored blueviolet). However, the additional bulkiness of the Trp contributes in expanding its to Phe541 and Phe513 also leading to the small shift in the alkyl chain of Arg530.
The of the two evolved variants R12-2 (3gkl) and R12-13 (3gjn) is very similar. The variant R12-2 carries . In the bound wildtype Im9 (yellow) Glu41 makes a with the ColE9’s Lys97 (1bxi). While in the R12-13/ColE7 complex the closest ColE7 residues R12-13 Glu41 are Thr531 (3.37Å) and Lys528 (8.85Å) (3gjn). In the R12-2/ColE7 complex the ColE7 residue to R12-2 Gly41 is Thr531 (9.48Å) (3gkl).
