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

Sharabi O, Peleg Y, Mashiach E, Vardy E, Ashani Y, Silman I, Sussman JL, Shifman JM - Protein Eng. Des. Sel. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.

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.

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(A) The amino acid sequence of Fas, showing the four intra-chain disulphide bridges and the interfacial residues chosen for redesign. (B) The Fas/TcAChE binding interface before redesign; (C) The Fas/TcAChE binding interface after redesign. Fas (pink) sits at the entrance of the narrow gorge leading to the active site of TcAChE (gray). The side-chains on Fas and TcAChE selected for computational optimization are displayed as pink and blue sticks, respectively. The numbers are shown for the Fas residues for which affinity-enhancing mutations had been predicted.
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GZP045F1: (A) The amino acid sequence of Fas, showing the four intra-chain disulphide bridges and the interfacial residues chosen for redesign. (B) The Fas/TcAChE binding interface before redesign; (C) The Fas/TcAChE binding interface after redesign. Fas (pink) sits at the entrance of the narrow gorge leading to the active site of TcAChE (gray). The side-chains on Fas and TcAChE selected for computational optimization are displayed as pink and blue sticks, respectively. The numbers are shown for the Fas residues for which affinity-enhancing mutations had been predicted.

Mentions: AChE is a synaptic enzyme that terminates impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine (Zimmerman and Soreq, 2006). Fas is a snake venom polypeptide toxin that is a very powerful reversible inhibitor of AChE (Karlsson et al., 1985); it belongs to the family of three-finger toxins that share a common structural motif: a β-sheet core stabilized by four disulfide bridges and three protruding loops that resemble human fingers (Kini, 2002). The crystal structure of the Fas/AChE complex has been solved for Torpedo californica AChE (TcAChE), mouse AChE (mAChE) and human AChE (hAChE) (Bourne et al., 1995; Harel et al., 1995; Kryger et al., 2000). The three structures are almost super-imposable, with Fas binding at the peripheral anionic site of the enzyme, thus sealing the narrow gorge that leads to the active site (Fig. 1B). Complexes of Fas with mammalian AChEs display very high affinities (Kd values of 10−11–10−12 M) (Karlsson et al., 1985; Eastman et al., 1995; Radic et al., 1995). A weaker, yet still high affinity (Kd of 4 × 10−10 M) was reported for the complex between Fas and TcAChE (Weiner et al., 2009). The tight binding between these two proteins has been attributed to several factors, including a remarkable surface complementarity, a large hydrophobic surface burial accompanying binding and formation of several inter-molecular hydrogen bonds (Harel et al., 1995). In addition, electrostatic interactions between cationic residues on the interaction surface of Fas and anionic residues on the interaction surface of AChE play an important role in the binding process (Radic et al., 1997).


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

Sharabi O, Peleg Y, Mashiach E, Vardy E, Ashani Y, Silman I, Sussman JL, Shifman JM - Protein Eng. Des. Sel. (2009)

(A) The amino acid sequence of Fas, showing the four intra-chain disulphide bridges and the interfacial residues chosen for redesign. (B) The Fas/TcAChE binding interface before redesign; (C) The Fas/TcAChE binding interface after redesign. Fas (pink) sits at the entrance of the narrow gorge leading to the active site of TcAChE (gray). The side-chains on Fas and TcAChE selected for computational optimization are displayed as pink and blue sticks, respectively. The numbers are shown for the Fas residues for which affinity-enhancing mutations had been predicted.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2742391&req=5

GZP045F1: (A) The amino acid sequence of Fas, showing the four intra-chain disulphide bridges and the interfacial residues chosen for redesign. (B) The Fas/TcAChE binding interface before redesign; (C) The Fas/TcAChE binding interface after redesign. Fas (pink) sits at the entrance of the narrow gorge leading to the active site of TcAChE (gray). The side-chains on Fas and TcAChE selected for computational optimization are displayed as pink and blue sticks, respectively. The numbers are shown for the Fas residues for which affinity-enhancing mutations had been predicted.
Mentions: AChE is a synaptic enzyme that terminates impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine (Zimmerman and Soreq, 2006). Fas is a snake venom polypeptide toxin that is a very powerful reversible inhibitor of AChE (Karlsson et al., 1985); it belongs to the family of three-finger toxins that share a common structural motif: a β-sheet core stabilized by four disulfide bridges and three protruding loops that resemble human fingers (Kini, 2002). The crystal structure of the Fas/AChE complex has been solved for Torpedo californica AChE (TcAChE), mouse AChE (mAChE) and human AChE (hAChE) (Bourne et al., 1995; Harel et al., 1995; Kryger et al., 2000). The three structures are almost super-imposable, with Fas binding at the peripheral anionic site of the enzyme, thus sealing the narrow gorge that leads to the active site (Fig. 1B). Complexes of Fas with mammalian AChEs display very high affinities (Kd values of 10−11–10−12 M) (Karlsson et al., 1985; Eastman et al., 1995; Radic et al., 1995). A weaker, yet still high affinity (Kd of 4 × 10−10 M) was reported for the complex between Fas and TcAChE (Weiner et al., 2009). The tight binding between these two proteins has been attributed to several factors, including a remarkable surface complementarity, a large hydrophobic surface burial accompanying binding and formation of several inter-molecular hydrogen bonds (Harel et al., 1995). In addition, electrostatic interactions between cationic residues on the interaction surface of Fas and anionic residues on the interaction surface of AChE play an important role in the binding process (Radic et al., 1997).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.

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.

Show MeSH
Related in: MedlinePlus