Phthalocyanine tetrasulfonates bind to multiple sites on natively-folded prion protein

The phthalocyanine tetrasulfonates (PcTS), a class of cyclic tetrapyrroles, bind to the mammalian prion protein, PrP. Remarkably, they can act as anti-scrapie agents to prevent the formation and spread of infectious, misfolded PrP. While the effects of phthalocyanines on the diseased state have been investigated, the interaction between PcTS and PrP has not yet been extensively characterized. Here we use multiple, complementary assays (surface plasmon resonance, isothermal titration calorimetry, fluorescence correlation spectroscopy, and tryptophan fluorescence quenching) to characterize the binding of PcTS to natively-folded hamster PrP(90–232), in order to determine binding constants, ligand stoichiometry, influence of buffer ionic strength, and the effects of chelated metal ions. We found that binding strength depends strongly on chelated metal ions, with Al3 +-PcTS binding the weakest and free-base PcTS the strongest of the three types tested (Al3 +, Zn2 +, and free-base). Buffer ionic strength also affected the binding, with Kd increasing along with salt concentration. The binding isotherms indicated the presence of at least two different binding sites with micromolar affinities and a total stoichiometry of ~ 4–5 PcTS molecules per PrP molecule.

► FB–, Al3+– and Zn2+–PcTS bind PrPC(90–232) with low μM affinity. ► Al3+– and Zn2+–PcTS bind PrPC(90–232) with a stoichiometry of 4–5 ligands/protein. ► Increasing Kd correlates with decreased anti–scrapie potency. ► PcTS binding to PrP is likely driven by interactions with aromatic residues. ► Electrostatic interactions can modulate PcTS–PrPC binding affinity.