In Silico Design of Protein–Protein Interaction Libraries for Fasciculin-2 Variant Selection

The interaction between fasciculin-2 (Fas) and acetylcholinesterase (AChE) is key to the study of neurodegenerative diseases, such as Alzheimer's and other synaptic dysfunctions. This project uses structural bioinformatics to guide the design of a Fas mutant library, to be screened by Yeast Surface Display (YSD), aiming to identify less toxic variants. Using RosettaCommons, 31 interface residues (within 8 Å of AChE) were selected for mutagenesis from the crystal structure of the Fas-AChE complex (PDB 1B41). For each position, substitutions were simulated using FastDesign with the ref2015 energy function, generating 6,200 mutants. The results were evaluated using energetic and packing metrics, with PBEE estimating the binding free energies for each mutant. Two mutants were selected based on extreme ΔGbind values: one with highest and another with lowest predicted affinity. Both showed loss of hydrogen bonds at the interface and an increase in unsatisfied polar groups, suggesting a trade-off between affinity and structural quality. They share mutations at positions 4, 7, 8, 11, 29 and 47, suggesting these are structurally tolerated changes (hotspots). Mutant 2699 presents exclusive mutations at positions 6 (H→C), 27 (R→L), 32 (K→L) and 33 (M→W), while mutant 0924 differs at positions 6 (H→F), 33 (M→I) and 37 (R→M). Positions 34, 58 and 61, with negative ΔΔG, were not mutated, suggesting they are structurally critical. Comparison with Aizner et al. (2014) validated our findings: M33W was reported as unfolded (despite high affinity in Rosetta), and mutations at R27 were experimentally destabilizing, consistent with the loss of positive charge in both mutants. This approach enables the rational selection of targets for biophysical studies. These results support the use of computational mutagenesis combined with structure-based energetic evaluation as an effective strategy for the design of less toxic variants upon selecting a larger mutant library.