D90N and D90K mutations in the chaperonin OBP reduce its ability to induce fibrillation of the prion protein by decreasing ATPase activity.

Abstract

We have previously shown that, in the presence of ATP, phage chaperonins stimulate amyloid transformation of the prion protein and alpha-synuclein, while in the absence of ATP they prevent fibrillation of amyloidogenic proteins. To confirm the involvement of chaperonins in the transformation of amyloidogenic proteins in an ATP-dependent manner, we obtained mutant forms of the single-ring chaperonin OBP with amino acid substitutions at the ATP binding site. It was shown that the D90 amino acid residue in the highly conserved GDGTTT motif is involved in ATP binding. Point mutations of D90N and D90K significantly reduced ATPase activity and destabilised the structure of chaperonin. The mutant chaperonins showed chaperone-like activity in the presence of ATP, protecting phage endolysin from thermal aggregation, similar to the wild-type chaperonin OBP. Although both mutant chaperonins are able to induce the conversion of prion protein monomers into amyloid fibrils in the presence of ATP due to residual ATPase activity, they function much less efficiently than the wild-type chaperonin OBP, affecting the initial stage of fibrillation, nucleation. Thus, a correlation between ATPase activity of chaperonin and its inducing effect on amyloid protein fibrillation has been shown. In addition, the possibility of obtaining mutant forms of chaperonins devoid of ATPase activity and preventing fibrillation of amyloid proteins even in the presence of ATP has been demonstrated.