In the last years, it has been demonstrated a link between the overload of metal ions inside nervous system cells and the onset of severe neurodegenerative diseases. This prompted the investigation of the structural and functional properties of transporters responsible for the uptake and regulation of metal ions inside cells. In particular, members of the Nramp/SLC11 ("natural resistance-associated macrophage proteins"/"solute carrier 11") family of transporters tightly regulate the influx of divalent transition-metal ions across cellular membranes contributing to cellular homeostasis. In these secondary active transporters, the translocation of divalent transition metal ions from the extracellular matrix to the cellular cytoplasm is coupled to proton transport (symport). The co-transport is realized using an alternate access mechanism in which the protein is alternately open towards the extracellular matrix, ("outward facing conformation", OFC) or towards the cellular cytoplasm ("inward facing conformation", IFC).

Although a wide range of functional and structural studies provided fundamental insights on the proton-coupled transport, most mechanistic details are still unknown. As an example, the coordination sphere of the metal ion in the inward-facing conformation of the protein has only been partially resolved by crystallographic experiments and later attempts to provide comprehensive descriptions led to contradictory models. Furthermore, several experimental evidence proved that proton uniport in the absence of the metal ion, is also possible, suggesting an unusual symporter behaviour for Nramps, in which the two substrates are not tightly coupled. Despite the recent advances in the comprehension of Nramps proton transport, the underlying mechanistic details remain still ambiguous, as demonstrated by inconsistent mechanistic models proposed during the last years.

In this thesis, computational methods have been used in order to provide new molecular level insights in the proton-coupled divalent transition metal ions transport performed by Nramps. In particular, a combination of classical molecular dynamics (MD) and QM/MM ab initio MD methods have been applied in order to refine the partially resolved coordination sphere of Mn2+ in the active site of Staphylococcus Capitis Divalent Metal-ion Transporter (ScaDMT), representing the only available crystal structure of a substrate-bound member of the family in an IFC. This QM/MM multiscale approach, using an accurate quantum treatment of the active site and a classical treatment of the surroundings, allowed for state-of-the-art calculations of the entire system that led to the unequivocal identification of the metal's coordination sphere.

Furthermore, MD simulations have been performed for both the IFC ScaDMT and Eremococcus Coleocola DMT (EcoDMT), whose crystal structure has been obtained in OFC. Since experimental evidences proved that proton transport can occur even in the absence of the metal ion substrate, we performed simulations of both proteins in their apo forms. These simulations allowed us to characterize four amino acidic residues identified as likely key players in the proton transport process and to determine which role they assume in the process. Finally, we were able to identify the likely primary proton acceptor and proposed a potential route followed by protons in their influx toward the intracellular cytoplasm.