Accelerated Fibril Formation of α-Synuclein by an IF-Inserted F36V Mutant

Abstract

FK506 binding proteins (FKBPs) are molecular chaperones with the cis-trans peptidyl-prolyl isomerase (PPIase) activity, that are used to help nascent proteins fold correctly. 1 Some FKBPs, including FKBP12, FKBP38, FKBP52, FKBP65, Pin1, and cyclophilin A, have been shown to possess PPIase activity, and have also exhibited the potential to modulate alpha-synuclein (α-Syn) aggregation, which plays a crucial role in Parkinson’s Disease (PD). 2 Among these, FKBP12 is the most potent for stimulating the aggregation of α-Syn, as has been demonstrated in vitro 3 as well as in a neuronal cell culture model. 4 These findings led us to develop better FKBP12-derived chaperones that can more effectively regulate the aggregation of α-Syn as targets for PD treatment. With an aim to construct a more powerful molecular chaperone with a higher PPIase activity, we engineered the F36V system, which is similar to that reported by the Schmid group. 5,6 In a previous study, we successfully constructed an F36VIF hybrid by replacing the flap of F36V with an insertion-in-flap(IF) domain from sensitive-to-lysis D (SlyD) protein of Escherichia coli (E. coli). 7 The IF that was inserted was the 61- amino acid domain starting with AYG and ending with LKF that protrudes from a loop of the FKBP domain near the PPIase catalytic site, which is indicative of its hydrophobicity. The F36V mutant imparted a better model to fit ligands via this protruding structural part than FKBP12. 8 By exploiting the structural contact between them, the Wandless group established a regulation system in which the stability of a target protein fused to some mutants of F36V depends on the presence of a small molecule, such as Shield-1; 9 when fused with a destabilizing domain (DD), the target protein is believed be degraded either via a proteasome pathway or by another process that has not yet been identified. Applying the cleaning-up concept that uses a DD domain to degrade pre-formed aggregated proteins, which are found in many neurodegenerative diseases (NDDs), we targeted the development of an IF-domain-inserted F36V system to regulate amyloid fibril formation. Using this system, we envision that the regulation of amyloid fibril formation could be switched on and off by a small molecule. In our previous study, we reported that F36VIF with the His6-tag gene was obtained by site-directed mutagenesis from FKBPIF/pET28a plasmid. 5 The resulting F36VIF protein was successfully expressed in BL21(DE3) in the presence of IPTG and further separated using Ni 2+ -affinity chromatography. The chymotrypsin-coupled assays 10 showed that the PPIase activity of F36VIF (202% of control) was remarkably higher than that of F36V itself (128% of control). 6 It is worth noting that the PPIaes activity of F36VIF