How do intricate multi-residue features such as protein-protein interfaces evolve? To address this question, we evolved a new colicin-immunity binding interaction. We started with Im9, which inhibits its cognate DNase ColE9 at 10(-14) M affinity, and evolved it toward ColE7, which it inhibits promiscuously (Kd > 10(-8) M). Iterative rounds of random mutagenesis and selection toward higher affinity for ColE7, and selectivity (against ColE9 inhibition), led to an approximately 10(5)-fold increase in affinity and a 10(8)-fold increase in selectivity. Analysis of intermediates along the evolved variants revealed that changes in the binding configuration of the Im protein uncovered a latent set of interactions, thus providing the key to the rapid divergence of new Im7 variants. Overall, protein-protein interfaces seem to share the evolvability features of enzymes, that is, the exploitation of promiscuous interactions and alternative binding configurations via 'generalist' intermediates, and the key role of compensatory stabilizing mutations in facilitating the divergence of new functions.
Following evolutionary paths to protein-protein interactions with high affinity and selectivity., Levin KB, Dym O, Albeck S, Magdassi S, Keeble AH, Kleanthous C, Tawfik DS, Nat Struct Mol Biol. 2009 Oct;16(10):1049-55. Epub 2009 Sep 13. PMID:19749752
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
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 variantsR12-2 (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 Tyr55Trp mutation in the conserved region. Both Tyr55 in R12-2 and Trp55 in R12-13 could sustain the
hydrophobic core and create a hydrogen bond 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 hydrophobic interactions to Phe541 and Phe513 also leading to the small shift in the alkyl chain of Arg530.
The overall conformation of the two evolved variants R12-2 (3gkl) and R12-13 (3gjn) is very similar. The variant R12-2 carries mutation E41G. In the bound wildtype Im9 (yellow) Glu41 makes a salt bridge with the ColE9’s Lys97 (1bxi). While in the R12-13/ColE7 complex the closest ColE7 residuescontactingR12-13 Glu41 are Thr531 (3.37Å) and Lys528 (8.85Å) (3gjn). In the R12-2/ColE7 complex the closestColE7 residue to R12-2 Gly41 is Thr531 (9.48Å) (3gkl).
