Identifying the copper coordination environment between interacting neurodegenerative proteins: A new approach using pulsed EPR with 14N/15N isotopic labeling

The trafficking and aggregation of neurodegenerative proteins often involve the interaction between intrinsically disordered domains, stabilized by the inclusion of physiological 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 (PrP^C) binds both monomers and aggregates of Alzheimer's amyloid-beta (Aβ), promoting Aβ internalization and aberrant signaling, respectively. Both proteins bind Cu²⁺ with high affinity, opening the potential for copper to form an intermolecular bridge. We describe here a novel approach utilizing multiple EPR experiments to investigate the simultaneous Cu²⁺ coordination of PrP^C and Aβ in a 1:1:1 mixture. Uniformly ¹⁵N-labeled PrP^C is used in conjunction with natural abundance ¹⁴N Aβ, the combination of which leads to distinct energy manifolds for paramagnetic Cu²⁺ and is resolved by the pulsed EPR experiments ESEEM and HYSCORE. We develop acquisition parameters to simultaneously optimize ¹⁴N (I = 1) and ¹⁵N (I = ½) pulsed EPR signals and we also advance the theory of ESEEM and HYSCORE to quantitatively describe multiple ¹⁵N imidazole coordination. This unique approach provides compelling evidence of a copper-stabilized ternary complex, with equatorial Cu²⁺ coordination formed by one histidine imidazole from Aβ and three from PrP. Moreover, the methodologies developed here provide a framework for assessing the copper environment in other interacting neurodegenerative proteins.