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

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.

Tradeoff Between Stability and Multispecificity in the Design of Promiscuous Proteins

“…Our WT recovery rates for single-state CaM designs are very similar to those observed, on average, when redesigning protein cores (51%) [24] and somewhat lower than that observed in our previous study, where the interface of a very high-affinity protein-protein complex was redesigned (62%) [25]. These results are reasonable, since CaM interactions with its targets are mostly conveyed by buried residues; the affinities of CaM-target complexes, while high, are not among the highest measured in nature.…”

What Makes Ras an Efficient Molecular Switch: A Computational, Biophysical, and Structural Study of Ras-GDP Interactions with Mutants of Raf

“…48,49 The difficulty in our case is augmented by the absence of the X-ray structure for the Ras GDP ·Raf WT complex; our model for such a structure is bound to introduce additional uncertainties into the design procedure. We expected that some of our designed mutations would result in reduced binding of Raf to Ras GDP in contrary to our predictions.…”

Increasing Affinity of Interferon-γReceptor 1 to Interferon-γby Computer-Aided Design

“…Interestingly, this study reports high cooperativity among the single mutations as the affinity of double mutants has been reported up to a thousand-times higher compared to WT. Computer model of binding between acetylcholine esterase and its inhibitor fasciculin [48] has predicted that increase of affinity can be achieved by mutating five interface fasciculin residues. However, to achieve a better binding, at least one of the five mutations had to be scrapped and actually the tightest interaction (sevenfold increase) occurred with just one of the originally designed mutations.…”