Following evolutionary paths to protein-protein interactions with high affinity and selectivity

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

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 (Kd ≤ 10-14 M) and selectivity(non-cognate binding is 106-1010-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 (> 1010 micro-droplets in 1 ml of oil). • The droplets are cell-free extractsof approximately 2 µm diameter. • About one gene per droplet, together with inactive ColE7 (activated by Co+2). • Selection: From Ref. 15 • Generate around 1010Im 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).

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:

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

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.