Mutant immunity protein 9 (variant R12-13)

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.

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).