The structural flexibility of the human copper chaperone Atox1: Insights from combined pulsed EPR studies and computations

Abstract

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.

The pivotal role of MBD4–ATP7B in the human Cu(i) excretion path as revealed by EPR experiments and all-atom simulations

“…Previously, by using various distance distribution constraints, we computed two distinct conformational states of the Atox1 homodimer, which we called closed and open conformations. 21 The distribution around 4.2 nm corresponds to a closed conformation, which agrees with that of PDB ID 3IWX, 51 while the distribution around 2.3 nm corresponds to an open conformation not yet trapped by either NMR or X-ray crystallography. 21 Interestingly, when adding spin-labelled Atox1 to MBD3-4 solution, the distribution around 4.2 nm disappears, suggesting that in the presence of MBD3-4, Atox1 no longer exists as a homodimer.…”

“…Consistently, the flexibility of b4 also increases upon Cu(I) binding to Atox1. 21 Continuous-wave (CW) EPR experiments support pulsed EPR data, and detect large amplitude motions for the MBD3-4_C305A mutant upon Cu(I) binding ( Table S1, Fig. S2 and S3, ESI †).…”

“…Atox1 expression, purification and spin labeling is similar to the expression of ATP7B and was described in a series of publications. [21][22][23]…”

“…This technique complements NMR and smFRET studies, owing to its sensitivity to the proteins' conformational flexibility in solution. For example, EPR experiments recently showed that Atox1 can accommodate distinct conformations, depending on the interacting partner protein, [21][22][23] and that it interacts as a homodimer with the Ctr1 intracellular domains. Conversely, allatom simulations have the potential to rationalize at the atomiclevel resolution the spectroscopic findings.…”

A novel function of M. tuberculosis chaperonin paralog GroEL1 in copper homeostasis

“…Copper chaperones play important role in shuttling metal ions to target proteins. The structure of metallochaperones must be sufficiently flexible both to carry specific ions while cytoplasmic movement and to transfer the ions to or from a partner protein (39). SAXS studies on GroEL1 protein show that GroEL1 protein is flexible and binding of copper (Cu 2+ ) ions causes conformational change in GroEL1 as well as increases its flexibility.…”