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

Abstract

Atox1–MBD4 interaction mediates the in-cell Cu(i) concentration.

An EPR Study on the Interaction between the Cu(I) Metal Binding Domains of ATP7B and the Atox1 Metallochaperone

“…Since the dynamics of a spin label bound to the C41 (C41R1) of Atox1 is relatively high, the only explanation for this is that when spin-labeled Atox1 interacts with MBD1-3 and MBD4-6, the dynamics of C41R1 is reduced, meaning that Atox1 is trapped between the various MBDs. This is in agreement with our previous findings [23], where we suggested that the Atox1 monomer interacts with MBD3-4 only in a face-to-face manner with MBD4, and that it is not affected by the presence of MBD3. Here, the dynamics of the spin-labeled C41R1 position of Atox1 are not affected and not reduced, since there is no other MBD nearby.…”

“…Figure 4 shows the DEER distance distribution functions obtained for the various MBDs in the absence and presence of Atox1. DEER experiments performed on MBD3-4 and MBD3-4_C305A in the presence of sl-Atox1 were already reported by us earlier, and showed a similar broad distance distribution function of between 1.5 and 3.5 nm [23], suggesting that Atox1 interacts only with MBD4, and that no conformational changes in MBD3 occurred while interacting with MBD4. MBD4-6, labeled at C358R1, C431R1 from MBD4, and C490R1 from MBD5 showed three distance distribution functions.…”

“…The CW-EPR spectrum of MBD3-4 is very similar to the CW-EPR spectrum of Atox1 dimer; it was also simulated with a dominant species (98%) and with a correlation time of 2•10 −9 s, and an isotropic electron-electron interaction (ω ee ) value of 5 MHz, where 2% of the spin-labels were simulated without an isotropic electron-electron interaction, but with the same dynamics. The addition of Cu(I) to MBD3-4 solution results in a spectrum with slightly higher dynamics, and a reduction in the isotropic electron-electron interaction, as reported before [23]. The addition of sl-Atox1 to MBD3-4 in the presence of Cu(I) did not affect the CW-EPR spectrum.…”

“…However, not all MBDs are equivalent in terms of receiving Cu(I) from Atox1. Magnetic resonance spectroscopy and computation studies demonstrated that Atox1 can form a stable adduct with MBD1, MBD2 and MBD4, which are more solvent-exposed; however, not with MBD3 and MBD5-6 [12,[21][22][23]. Although MBD5-6 are critical domains for proper copper transport to the membrane domain [24], it is still unclear how copper is transferred to these domains.…”

“…Recently, we conducted electron paramagnetic resonance (EPR) spectroscopy experiments in order to better understand the interaction between the Atox1 metallochaperone and MBD3-4 as a function of Cu(I) binding [23,25]. The power of EPR spectroscopy lies in its ability to target conformational changes in solution, without limiting the protein size or the system.…”