UC Santa Cruz

Investigating the Cellular Prion Protein’s Metal Driven cis Interaction

Kathleen A. Markham

The cellular prion protein (PrPC) undergoes an N-C interdomain interaction upon the addition of endogenous metal ligands. Studies demonstrated that the octarepeat domain coordinates copper or zinc, which elicits this interdomain contact termed cis interaction. What is unclear is if physiologically relevant N-terminal segments are assisting in PrPC’s cis interaction. These N-terminal segments have been shown to play a potentially significant role in the function and regulation of the cellular prion protein. While full knockdown of PrPC in mice is relatively benign, the deletion of an internal 21 amino acid stretch, known as ΔCR, produces neonatal lethal toxicity. The polybasic tail, 23KKRPKPGGW31, has been shown to associate with ionotropic membrane receptors and is essential for ΔCR toxicity. Through the use of 15N-1H HSQC NMR, LC-MS/MS, and electrophysiology studies, our findings indicated that both the polybasic tail and central region are essential components in assisting the cis interaction. Additionally, elimination of the positive charge on the polybasic tail reduces the measured cis interaction, which indicates that the cis interaction is partially assisted through electrostatics. Furthermore, 113Cd, 2JNH, and 15N-1H HSQC NMR were employed to investigate PrPC’s octarepeat domain when complexed with diamagnetic metal ions. These results provided detailed information about the coordination sphere for Zn2+ that had been previously lacking. Multi-dimensional NMR findings showed that Cd2+ induced a cis interaction and that coordination was localized to the most distal epsilon nitrogen on the histidine imidazole ring within the octarepeat domain. 113Cd NMR was able to successfully measure PrPC’s cis interaction and any weakened interactions resulting from pathological mutations. Together, these results demonstrate that physiologically relevant segments of PrPC participate in the cis interaction and misregulation could lead to toxicity. Additionally, we have demonstrated that Cd2+ via 113Cd NMR is a viable surrogate for measuring Zn2+’s driven cis interaction