UC Santa Cruz

The cellular prion protein (PrPC) is a membrane-anchored glycoprotein consisting of two domains: a flexible N-terminal domain that participates in metal binding, and a mainly helical C-terminal domain that converts to β-sheet structure in the course of prion disease. These two domains have traditionally been thought of as non-interacting; however, recent cellular and biophysical evidence has forced a reconsideration of this view. Here we employ nuclear magnetic resonance spectroscopy (NMR), electron paramagnetic resonance spectroscopy (EPR), and fluorescence resonance energy transfer (FRET) to investigate inter-domain structure in PrPC in response to metal binding. We develop a new strategy for probing domain-domain interactions in PrP that employs the genetic incorporation and labeling of the unnatural amino acid pAcPhe. Through 1H-15N HSQC NMR experiments on full-length and truncated PrP constructs, we demonstrate a novel tertiary fold in which the Zn2+-bound octarepeat domain contacts the exposed surfaces of helices 2 and 3. These results are supported by inter-domain distance measurements from EPR and FRET. The apparent stability of this interaction is diminished in several mutant PrPs that result in familial prion disease, suggesting a potential role for inter-domain structure in disease progression. We then examine global structure in Cu2+-bound PrPC using site-directed spin labeling and EPR spectroscopy. Distance measurements between Cu2+, bound with high affinity to the octarepeat domain, and spin labeled residues of the globular C-terminus reveal that the copper-bound octarepeats interacts with a negatively charged surface defined by helices 2 and 3. This interaction surface is qualitatively equivalent to the tertiary contact site identified in the Zn2+-bound protein. Our results are supported by molecular dynamics simulations and indicate that this cis interaction is stabilized by electrostatics. Our findings suggest that metal-induced tertiary structure may be a general property of PrPC, and that disruption of this interaction may be a contributing factor in prion disease pathology.