Identifying the Copper Coordination Environment Between Interacting Neurodegenerative Proteins: A New Approach Using Pulsed EPR with 14N/15N Isotopic Labelling
The trafficking and aggregation of neurodegenerative proteins often involves the interaction between intrinsically disordered domains, stabilized by the inclusion of physiologic metal ions such as copper or zinc. Characterizing the metal ion coordination environment is critical for assessing the stability and organization of these relevant protein-protein interactions but is challenging given the lack of regular molecular order or global structure. The cellular prion protein (PrPC) binds both monomers and aggregates of the Alzheimers amyloid-beta peptide (A{beta}), promoting interactions of relevance to A{beta} internalization across the cellular plasma membrane and aberrant signaling in neurodegenerative disease, respectively. Both proteins bind Cu2+ with high affinity, suggesting the existence of a ternary complex with copper bridging between the two proteins through His coordination. In this work, we describe a novel approach utilizing multiple EPR experiments to characterize the simultaneous Cu2+ coordination of PrPC and A{beta}. Uniformly 15N-labeled PrPC is used in conjunction with natural abundance 14N A{beta}, the combination of which leads to distinct energy manifolds for paramagnetic Cu2+ and resolved by the pulsed EPR experiments ESEEM and HYSCORE. We develop acquisition parameters to simultaneously optimize 14N (I = 1) and 15N (I = [1/2]) pulsed EPR signals and we also advance the theory of ESEEM and HYSCORE to quantitatively describe multiple 15N imidazole coordination. Together, these findings provide a detailed view of how Cu2+ bridges between the two proteins in this complex, along with a global strategy for assessing the copper environment with other interacting neurodegenerative proteins.