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Following evolutionary paths to protein-protein interactions with high affinity and selectivity Levin KB, Dym O, Albeck S, Magdassi S, Keeble AH, Kleanthous.

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Presentation on theme: "Following evolutionary paths to protein-protein interactions with high affinity and selectivity Levin KB, Dym O, Albeck S, Magdassi S, Keeble AH, Kleanthous."— Presentation transcript:

1 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: (2009)

2 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 (K d ≤ M) and selectivity (non-cognate binding is fold weaker than cognate binding).

3 Goal of the experiment Begin with wild-type Im9 (Im9 inhibits ColE9 but not ColE7) Evolve it toward the inhibition of ColE7 while against ColE9 inhibition.

4 Experimental method Water-in-oil emulsion (> 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 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

5 Experimental procedure 8 rounds of mutation & selection with ColE7 (gradually increase selection pressure by increasing ColE7 concentration) 3 rounds of mutation & dual selection with ColE7 and 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)

6 Representative Im variants:

7

8 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

9 “Dual recognition” hypothesis Conserved hotspot Conserved throughout the family, serve as a common anchoring and starting point. Variable region Not in direct contact with ColE7, but results in different residues making the contact, thus mediate specificity. Binding configurations of Im9, R12-2, and Im7.

10 Interactions with ColE7: a)Im9 b)Im7 c)R12-2 New binding specificity occurred primarily by exploitation of latent interactions, without changing the sequence of the Im-contacting residues.

11 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.

12 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.


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