Molecular dynamics simulations in: High-resolution structures with bound Mn2+ and Cd2+ map the metal import pathway in an Nramp transporter

Transporters of the Nramp (Natural resistance-associated macrophage protein) family import divalent transition metal ions into cells of most organisms. By supporting metal homeostasis, Nramps prevent disorders related to metal insufficiency or overload. Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site. We present high- resolution structures of Deinococcus radiodurans Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate, Mn2+, across conformations and conserved networks of polar residues lining the inner and outer gates. A Cd2+-bound structure highlights differences in coordination geometry for Mn2+ and Cd2+. Measurements of metal binding using isothermal titration calorimetry indicate that the thermodynamic landscape for binding and transporting physiological metals like Mn2+ is different and more robust to per..., Molecular dynamics (MD) simulations were initialized from three high-resolution structures of DraNramp: the outward-open G223W•Mn2+ structure (6BU5) with Mn2+ removed and W223 mutated back to the native glycine residue in silico, the inward-open WT•Cd2+ structure with Cd2+ removed, and the inward-occluded WT structure. Crystallographic waters were retained, and protonation states of key titratable residues were selected with PROPKA (M. H. Olsson, Sondergaard, Rostkowski, & Jensen, 2011; Sondergaard, Olsson, Rostkowski, & Jensen, 2011) assuming a pH of 5.0 for residues exposed to external solvent and a pH of 7.0 for residues exposed to cytosol, a condition under which DraNramp exhibits high activity. All structures were oriented in the membrane with the PPM web server and membrane systems were prepared with CHARMM-GUI (Jo, Kim, Iyer, & Im, 2008; Lee et al., 2016). A POPC membrane of surface area 99 x 99 Å was constructed in the XY plane around the protein (Wu et al., 2014), t..., Can be visualized in VMD or PyMOL, but files are likely too large and should be either strided first (e.g. with catdcd) or analyzed directly, e.g. in Python, likely on a supercomputing cluster. If you want to use these simulations but do not have access to sufficient computing resources, feel free to reach out to us directly. See https://github.com/samberry19/nramp-md for how these simulations are analyzed in the manuscript.