Abstract
In-cell electron paramagnetic resonance (EPR) spectroscopy experiments provide high-resolution data about conformational changes of proteins within the cell. However, one of the limitations of EPR is the requisite of stable paramagnetic centers in a reducing environment. We recently showed that histidine-rich sites in proteins hold a high affinity to Cu(II) ions complexed with a chelator. Using a chelator prevents the reduction of Cu(II) ions. Moreover, this spin-labeling methodology can be performed within the native cellular environment on any overexpressed protein without protein purification and delivery to the cell. Herein, we use this novel methodology to gain spatial information on the extracellular domain of the human copper transporter, hCtr1. Limited structural information on the transmembrane domain of the human Ctr1 (hCtr1) was obtained using X-ray crystallography and cryo-EM. However, these structures are missing information on the disordered extracellular domains of hCtr1. Extracellular domains are sensing or interacting with the environment outside of the cell and therefore play an essential role in any transmembrane protein. Especially in hCtr1, the extracellular domain functions as a gating mechanism for copper ions. Here, we performed EPR experiments revealing structural information about the extracellular N-terminal domain of the full-length hCtr1 in vitro and in situ in insect cells and cell membrane fragments. The comparison revealed that the extracellular domains of the in situ and native membrane hCtr1 are further apart than the structure of the purified protein. These method-related differences highlight the significance of studying membrane proteins in their native environment.