Key role of methionine-rich domains in copper homeostasis systems: focus on CueOs – MetCop

Copper element is at the same time an essential micronutrient in living systems, being a cofactor of enzymes involved in many biological processes, but toxic for the cell when in excess. The understanding of mechanisms involved in intracellular copper balance between requirement and toxicity is thus crucial from fundamental and applicative points of view.
Among amino acids, methionine (Met) residues readily bind copper, and interestingly many proteins involved in copper homeostasis are rich in Met. As a model case, Escherichia coli CueO multi-copper oxidase, a periplasmic protein early recognised to be involved in copper resistance in vivo, presents a Met-rich domain proposed to be involved in the oxidation of Cu+ into the less toxic Cu2+. Intriguingly, such regions in CueOs greatly differ in term of structure and Met content according to the microorganisms.
The MetCop project aims at determining the role of CueO Met-rich domains in the mechanisms selected by various microorganisms along evolution to tackle copper stress. Two fundamental questions related to their involvement in copper homeostasis will be addressed: 1) To which extent Met-rich domains govern cuprous oxidase activity, hence copper detoxification? 2) What are the consequences of Met oxidation in Met-rich domains on copper tolerance? Through a multiscale and multidisciplinary approach, the MetCop project will allow to establish a clear correlation between i) structural features of Met-rich domains of a library of CueOs, ii) copper binding properties, iii) in vitro enzymatic activity and iv) in vivo copper resistance.
The multiscale approach set in the MetCop project involves entire cells, periplasmic extracts, purified proteins and synthetic peptide models, allowing a back and forth iterative process between cellular and molecular scales to answer the two fundamental targeted questions. More specifically, the molecular basis of Cu binding to pseudopeptides designed from identified Met-rich domains of CueOs, will improve our knowledge on copper-Met coordination and guide mutation of specific Met in the Met-rich domains of CueOs from various microorganisms. Changes in cuprous oxidase activities as a function of Met-rich domain structural features will be correlated to in vivo copper resistance. MetCop thus ambitions to precisely determine how and which Met(s) in Met-rich domains are involved in copper binding and copper resistance. Beyond the involvement in cuprous oxidase activity, MetCop will search for a role of Met(s) in Met-rich domains in the protection against reactive oxygen species that can be produced in the presence of copper. It will finally investigate whether MsrP, a protein recognized to reduce oxidized Met(s), may participate to maintain CueO activities in vivo.
The multidisciplinary approach is reflected by the panel of methodologies (some of them being especially set up in the MetCop project) carried out to determine the role of Met-rich domains of CueOs from different organisms in copper resistance: molecular biology, bioinformatics, biochemistry, chemical pseudopeptide design, theoretical methods and electrochemistry. This multidisciplinary approach is allowed thanks to a unique consortium with complementary expertise in i) genetics to study in vivo copper tolerance (LCB), ii) biophysics and biochemistry of redox enzymes including Cu-based metalloproteins (BIP), and iii) chemistry of copper binding to peptides (SYMMES).
Such a multiscale, multidisciplinary approach is expected to provide new findings that will pave the way to the understanding of the evolutive selection of Met residues at specific position within proteins involved in copper homeostasis.