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My PhD thesis is entitled "Structure, molecular dynamics and selectivity of copper and mercury metallochaperones" and was carried out at the French Commissariat à l'Énergie Atomique(CEA), Grenoble (France). The project was part of a French national project that aimed at improving the knowledge related to nuclear toxicology (Programme de Toxicologie Nucléaire, ToxNuc). This initiative still exists and has been extented to nuclear and environmental toxicology (Programme de Toxicologie Nucléaire et Environnementale, ToxNuc-E). During my thesis, I focussed on the study of metallochaperones which are metal-binding proteins that convey metal ions from their plasma membrane transporter(s) to their cytosolic target(s). Specifically, I studied the yeast Atx1 and the human Hah1 which are both Cu(I) transporters and the bacterial MerP which carries Hg(II). The approach was two-fold and mainly computational:

  • molecular dynamics study of these 3 metallochaperones in their apo and holo states (the Cu(I)- and Hg(II)-bound states
  • accurate determination of the local structure of the metal-binding site of CuAtx1 in various conditions with X-ray absorption spectroscopy (XANES and EXAFS)

The thesis can be downloaded. It is in French and entitled: "Structure, dynamique moléculaire et sélectivité de métallochaperones à cuivre et à mercure".

abstract

Copper metallochaperones ensure delivery of Cu(I) ions towards target proteins in the cell. Metallochaperones of the Atx1 family exhibit a high sequence homology and the same fold. MerP, a mercury metallochaperone, share the same feature of sequence and structure. This thesis aims at underlining some dynamical and structural properties responsible for selectivity of metallochaperones for Cu(I) and Hg(II).

Molecular dynamics simulations of copper metallochaperones, Atx1 and Hah1, and of MerP, in the apo forms, and bound to Cu(I) or Hg(II), revealed dynamical and energetic characteristics common to the three metallochaperones when they chelate their native metal. An interaction network between the metal-binding site loop and two other loops around the site, was identified and varies from a metallated state to another. Thus, it could define a probable selectivity for metals. The loop of the chelation site shows a great structuration in presence of metal, accompanied by a rigidifyinf effect if this metal is native.

X-ray absorption experiments of Cu(I) bound by Atx1 showed that Cu(I) always has a trigonal geometry whose ligands are the cysteines of the metal-binding site, and an endogenous or exogenous ligand. So Atx1 always offers Cu(I) its preferential geometry. This property is a determinant of the selectivity of Cu(I) with respect to other metals.

In presence of glutathione, the Atx1-Cu(I) complex forms a binuclear homodimer associated to two glutathione molecules. Involvement of the exogenous ligand is suggested as an in vivo selectivity factor of Atx1 for Cu(I), and could favor the recognition by Atx1 of its target protein.

The molecular dynamics simulations were run with CHARMM (with the all-atom CHARMM27 forcefield). The X-ray absorption spectroscopy experiments (XANES and EXAFS) of Cu(I)-bound Atx1 were carried out at the European Synchrotron Radiation Facility (ESRF) in Grenoble at the BM30B/FAME beamline in close collaboration with Dr C. Den Auwer.

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