Insertion of IF (insertion-in-flap) Domain into F36V to Provide Higher Activity of Peptidyl-propyl cis-trans Isomerase

Abstract

Molecular chaperones are required to help nascent proteins fold properly or protect cells against various stresses by means of preventing their aggregation. Attempts to elucidate the molecular mechanisms by which chaperones can solubilize aggregation-prone proteins such as Aβ and αsynuclein (α-Syn) are relatively limited. So far, just a few publications have been written on the functional roles of chaperones such as Hsp104 and FKBP12 on controlling the aggregation of α-Syn into fibrillar structures. In our previous study, we described how functional Hsp104 protein not only inhibited α-Syn aggregation but also resolubilized aggregated protein in vitro. Recently, Engelborghs group has reported that FKBP12, containing the peptidyl-prolyl isomerase (PPIase) activity, accelerated fibril formation of α-Syn containing 5 Pro residues, which can be isomerized between cisand trans-conformations. Little is known about the molecular details that explain how molecular chaperones regulate fibril formation in many neurodegenerative diseases such as Alzheimer disease and Parkinson disease. In pursuit of developing a candidate regulator of fibril formation, we have attempted to engineer the FKBP system to acquire a higher activity of PPIase along with the chaperone activity. The IF (insertion-in-flap) domain with 61amino acids starting with AYG and ending with LKF was adopted from SlyD (sensitive-to-lysis) in Escherichia coli (E. coli) as well as from MtFKBP17 of Methanococcus thermolithotrophicus. The structural analysis of the IF domain, protruding from a loop of the FKBP domain near the PPIase catalytic site, indicated its hydrophobicity so that it can interact with unfolded proteins in the nonpolar region found in extended structures such as a β-sheet. Folding of the IF domain is influenced by the FKBP domain, in such a way that the guest IF domain of SlyD folds rapidly when the host FKBP domain of SlyD is folded and is unfolded when the host is unfolded. Construction of an FKBPIF hybrid replacing the flap by an IF domain was previously reported by the Schmid group. The resulting hybrid FKBPIF protein exhibited strong folding activity as well as chaperone activity. For example, upon the refolding of RCM-T1 (a disulfide-reduced and Scarboxymethylated form of a variant of RNase T1), the hybrid FKBPIF showed the kcat/Km enhanced by 200 fold. In monitoring chaperone activity, FKBPIF exhibited inhibition of CS (citrate synthase) aggregation whereas FKBP did not. The insertion effect of IF in FKBPIF was also confirmed in the inhibited aggregation of insulin reduced by dithiothreitol. Upon structural analysis of the binding site of FKBP for FK506, it was envisioned that it would be surrounded by hydrophobic residues such as F36. Further mutation of F36 to V36 yielded a better system in terms of fitting ligands through a protruding structural part. Using the structural contact between them, the Wandless group have developed a regulation system in which the stability of a certain domain