CueR (Cu export regulator) is a metalloregulator protein that "senses" Cu(I) ions with very high affinity, thereby stimulating DNA binding and the transcription activation of two other metalloregulator proteins. The crystal structures of CueR when unbound or bound to DNA and a metal ion are very similar to each other, and the role of CueR and Cu(I) in initiating the transcription has not been fully understood yet. Using double electron-electron resonance (DEER) measurements and structure modeling, we investigate the conformational changes that CueR undergoes upon binding Cu(I) and DNA in solution. We observe three distinct conformations, corresponding to apo-CueR, DNA-bound CueR in the absence of Cu(I) (the "repression" state), and CueR-Cu(I)-DNA (the "activation" state). We propose a detailed structural mechanism underlying CueR's regulation of the transcription process. The mechanism explicitly shows the dependence of CueR activity on copper, thereby revealing the important negative feedback mechanism essential for regulating the intracellular copper concentration.

Metallochaperones are responsible for shuttling metal ions to target proteins. Thus, a metallochaperone's structure must be sufficiently flexible both to hold onto its ion while traversing the cytoplasm and to transfer the ion to or from a partner protein. Here, we sought to shed light on the structure of Atox1, a metallochaperone involved in the human copper regulation system. Atox1 shuttles copper ions from the main copper transporter, Ctr1, to the ATP7b transporter in the Golgi apparatus. Conventional biophysical tools such as X-ray or NMR cannot always target the various conformational states of metallochaperones, owing to a requirement for crystallography or low sensitivity and resolution. Electron paramagnetic resonance (EPR) spectroscopy has recently emerged as a powerful tool for resolving biological reactions and mechanisms in solution. When coupled with computational methods, EPR with site-directed spin labeling and nanoscale distance measurements can provide structural information on a protein or protein complex in solution. We use these methods to show that Atox1 can accommodate at least four different conformations in the apo state (unbound to copper), and two different conformations in the holo state (bound to copper). We also demonstrate that the structure of Atox1 in the holo form is more compact than in the apo form. Our data provide insight regarding the structural mechanisms through which Atox1 can fulfill its dual role of copper binding and transfer. Statement (50-75 Words)Using EPR spectroscopy and computations, we show that the human copper metallochaperone Atox1 can accommodate at least six different conformational states. We also show that, upon binding Cu(I), the metallochaperone's structure becomes more compact and rigid. This research highlights how the structure of Atox1 enables it to fulfill its dual role of Cu(I) coordination and transfer, and points to the importance of the flexibility of this structure in controlling the copper transfer mechanism.