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

Paper presentation by Jintao Liu (10/13/2009)

Nat Struct Mol Biol, 16:1049-1055 (2009)

Introduction• Study members of the colicin-immunity family (ColE7-Im7 and ColE9-Im9).

• Colicin endonucleases (ColE) are used by E. coli to kill competing bacterial strains under stress conditions.

• The immunity (Im) proteins provide protection to the attacking bacteria from destruction of their own DNA.

• The cognate pairs bind with extremely high affinity (Kd ≤ 10-14 M) and selectivity (non-cognate binding is 106-1010-fold weaker than cognate binding).

Goal of the experiment

Begin with wild-type Im9(Im9 inhibits ColE9 but not ColE7)

Evolve it toward the inhibition of ColE7while against ColE9 inhibition.

Experimental method• Water-in-oil emulsion (> 1010 micro-droplets in 1 ml of oil).• The droplets are cell-free extracts of approximately 2 µm diameter.• About one gene per droplet, together with inactive ColE7 (activated by Co+2).

• Selection:

• Generate around 1010 Im variants before each round of selection.• Mutation: Amplify the survived genes with error-prone PCR (polymerase

chain reaction, about 3 or 4 mutations per gene).

From Ref. 15

Experimental procedure

8 rounds of mutation & selection with ColE7(gradually increase selection pressure by

increasing ColE7 concentration)

3 rounds of mutation & dual selection with ColE7and large excess of the ColE9 H103A mutant

1 round of in vivo screening(measure the highest ColE7/ColE9 concentration

the Im variants can protect against)

Representative Im variants:

Crystal structures

• ColE9 + Im9 (1BXI)

• ColE9 + Im9 E41A (1FR2)

• ColE7 + Im7 (7CEI)

• ColE7 + R12-2 (3GKL)

• ColE7 + R12-13 (3GJN)

• No crystal for round 8

“Dual recognition” hypothesis

• Conserved hotspotConserved throughout the family, serve as a common anchoring and starting point.

• Variable regionNot in direct contact with ColE7, but results in different residues making the contact, thus mediate specificity.Binding configurations of Im9, R12-2, and Im7.

Interactions with ColE7:

a) Im9b) Im7c) R12-2

New binding specificity occurred primarily by exploitation of latent interactions, without changing the sequence of the Im-contacting residues.

• Early mutations occurred in noncontacting residues.

• Reminiscent of the ‘evolution’ of antibody responses, in which affinity maturation is often mediated by mutations in noncontacting residues that fix the conformation of the binding loops.

• Many mutations that induce changes in enzyme specificity occur in second-shell residues and affect the conformation of active site loops.

• The mutations led to loss of stability, which reduce the level of soluble, functional inhibitors. (urea denaturation measurements)

• Mutation V37I enable the regain of stability.

• It seems that mutations that endow new functions are generally destabilizing, and stabilizing, compensatory mutations constitute a key step in the evolutionary trajectories of all types of protein functions.