Tracking Disordered\nExtracellular Domains of Membrane\nProteins in the Cell with Cu(II)-Based Spin Labels

<i>In-cell</i> electron paramagnetic resonance\n(EPR)\nspectroscopy experiments provide high-resolution data about conformational\nchanges of proteins within the cell. However, one of the limitations\nof EPR is the requisite of stable paramagnetic centers in a reducing\nenvironment. We recently showed that histidine-rich sites in proteins\nhold a high affinity to Cu(II) ions complexed with a chelator. Using\na chelator prevents the reduction of Cu(II) ions. Moreover, this spin-labeling\nmethodology can be performed within the native cellular environment\non any overexpressed protein without protein purification and delivery\nto the cell. Herein, we use this novel methodology to gain spatial\ninformation on the extracellular domain of the human copper transporter,\nhCtr1. Limited structural information on the transmembrane domain\nof the human Ctr1 (hCtr1) was obtained using X-ray crystallography\nand cryo-EM. However, these structures are missing information on\nthe disordered extracellular domains of hCtr1. Extracellular domains\nare sensing or interacting with the environment outside of the cell\nand therefore play an essential role in any transmembrane protein.\nEspecially in hCtr1, the extracellular domain functions as a gating\nmechanism for copper ions. Here, we performed EPR experiments revealing\nstructural information about the extracellular N-terminal domain of\nthe full-length hCtr1 in vitro and in situ in insect cells and cell\nmembrane fragments. The comparison revealed that the extracellular\ndomains of the in situ and native membrane hCtr1 are further apart\nthan the structure of the purified protein. These method-related differences\nhighlight the significance of studying membrane proteins in their\nnative environment.