Anti-Prion Ligand Binding Promotes Native PrP Folding Over Misfolding at the Single Molecule Level

Abstract

Ligands that bind to the prion protein, PrP, and prevent the spread of the diseased state have been discovered, but their mechanism of action remains uncertain. Determining anti-prion mechanisms may provide insight into the still-unknown means by which native PrP is converted to the infectious form. To explore the effects of an anti-prion ligand at the molecular level, we used force spectroscopy to study how a tetrapyrrole known to have anti-prion activity, iron(III)meso-tetra(N-methyl-4-pyridyl-prophine) or Fe-TMPyP, alters the folding behavior of individual PrP molecules. Single PrP molecules were unfolded using optical tweezers in the presence and absence of Fe-TMPyP. Ligand binding to the native structure was found to significantly increase its unfolding force. Not only did Fe-TMPyP binding stabilize the native state as expected from ensemble binding studies, but analysis of the unfolding force distributions revealed that Fe-TMPyP binding altered the nature of the transition state for unfolding the native structure: the energy barrier moved closer to the native state, making the transition state more compact, and the barrier height increased. Unexpectedly, Fe-TMPyP was also able to bind to PrP when it was unfolded or only partially folded, thereby delaying the normally rapid refolding into the native state. Probing the effects of Fe-TMPyP on inter-molecular interactions by measuring PrP dimers revealed that ligand binding promoted the formation of the native structure in individual monomers, preventing the formation of a thermodynamically-stable misfolded dimeric state. The ligand thus promotes native folding by stabilizing the native state while at the same time suppressing interactions that drive the formation of stable aggregates. These results suggest parallels between pharmacological chaperones like Fe-TMPyP and cellular chaperones.