(A) Electrostatic potential maps of HMA6 and HMA8 were modelled from the structure of LgCopA (PDB code 4BBJ) [11] with the MODELLER program [33]. The HMs are oriented with kinked helix III in front. The figure highlights charge differences between surface residues of HMA6 and HMA8 in the inner cell region. HMA6 presents positively charged residues (coloured blue) such as Lys³⁴¹ and Lys³⁴⁵ which align (both in sequence and in structure) with the negatively charged residues (coloured red) Glu²⁷³ and Asp²⁷⁷ of HMA8. Notice that the patch of residues Ser⁵³⁷ to Leu⁵⁴¹ in HMA6 exposes their backbone amine group (positive polarity) in a region of the sequence which structurally aligns with the negatively charged Asp⁴⁸⁰–Asp⁸⁵ region in HMA8. (B) Docking of PC [modelled from the structure of poplar PC (PDB code 4DP7) [34]] to HMA8 using the program HADDOCK [38]. Residues Cys⁸⁴ and His⁸⁷ in PETE2 and Asp²⁸², Met⁸⁵⁷ in HMA8 were defined as active residues for HADDOCK meaning that the program favours the presence of these residues at the docking interface. The proteins are shown in cartoon representation inside a phospholipid membrane. The Cu-binding site in PC constituted of amino acids His³⁷, Cys⁸⁴ and His⁸⁷ and a possible Cu route inside HMA8 leading from Cys⁵⁰⁴, Cys⁵⁰⁶ (CPC motif) to Met⁸⁵⁷ and Asp²⁸² are highlighted in CPK. (C) Electrostatic potential maps for PC and HMA8. Proteins were docked as shown in (B) then rotated so as to show their interface residues. Positively charged patches (coloured blue) appear in PC due to the presence of Lys³⁰ or Lys⁹⁵, for instance, whereas interfacial residues in HMA8 are mostly negatively charged (red) with lots of aspartic and glutamic acid residues (Asp²¹², Glu²⁷³, Glu²⁷⁵, Asp²⁷⁷, Asp⁴⁹⁸, Asp⁸⁴⁵…). Asp²⁸² in HMA8 and His⁸⁷ in PC proposed to bind Cu along its way outside the ATPase are shown in green CPK representation. Figures and molecular surfaces were drawn with program VMD [37] and electrostatic maps calculated with APBS [36] within VMD.