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.
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