Design, expression and characterization of mutants of fasciculin optimized for interaction with its target, acetylcholinesterase

Oz Sharabi, Yoav Peleg, Efrat Mashiach, Eyal Vardy, Yacov Ashani, Israel Simian, Joel L. Sussman, Julia M. Shifman

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

Predicting mutations that enhance protein-protein affinity remains a challenging task, especially for high-affinity complexes. To test our capability to improve the affinity of such complexes, we studied interaction of acetylcholinesterase with the snake toxin, fasciculin. Using the program ORBIT, we redesigned fasciculin's sequence to enhance its interactions with Torpedo californica acetylcholinesterase. Mutations were predicted in 5 out of 13 interfacial residues on fasciculin, preserving most of the polar inter-molecular contacts seen in the wild-type toxin/enzyme complex. To experimentally characterize fasciculin mutants, we developed an efficient strategy to over-express the toxin in Escherichia coli, followed by refolding to the native conformation. Despite our predictions, a designed quintuple fasciculin mutant displayed reduced affinity for the enzyme. However, removal of a single mutation in the designed sequence produced a quadruple mutant with improved affinity. Moreover, one designed mutation produced 7-fold enhancement in affinity for acetylcholinesterase. This led us to reassess our criteria for enhancing affinity of the toxin for the enzyme. We observed that the change in the predicted inter-molecular energy, rather than in the total energy, correlates well with the change in the experimental free energy of binding, and hence may serve as a criterion for enhancement of affinity in protein-protein complexes.

Original languageAmerican English
Pages (from-to)641-648
Number of pages8
JournalProtein Engineering, Design and Selection
Volume22
Issue number10
DOIs
StatePublished - Oct 2009

Bibliographical note

Funding Information:
This study was supported by the Israeli Ministry of Health (J.M.S), by the Israel Science Foundation and by the Divadol Foundation (J.L.S.) J.L.S. is the Morton and Gladys Pickman Professor of Structural Biology. This work was in collaboration with the Israel Structural Proteomics Center (ISPC), supported by The Israel Ministry of Science, Culture, and Sport, the Divadol Foundation, the Neuman Foundation and the European Commission Sixth Framework Research and Technological Development Program ‘SPINE2-COMPLEXES’ Project Contract No. 031220. Funding to pay the Open Access publication charges for this article was provided by the Divadol Foundation.

Keywords

  • Acetylcholinesterase/binding affinity/computational protein design/fasciculin/protein-protein interactions

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