DOI: 10.5061/dryad.tx95x6b2b
title: Molecular dynamics simulations in: High-resolution structures with bound Mn2+ and Cd2+ map the metal import pathway in an Nramp transporter

description: 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 perturbation than for
 transporting the toxic Cd2+ metal.
description: 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), the system was solvated in a 100 x 100 x 100 Å3
 rectangular box using TIP3 waters and electronically neutralized using
 potassium and chlorine ions at an overall concentration of 150 mM. The
 overall system size was approximately 103,000 atoms.
 All-atom simulations were run using GPU-accelerated NAMD
 (Phillips, Stone, & Schulten, 2008) and the CHARMM36m forcefield
 (Huang et al., 2017). Prior to simulation, the energy of each system was
 minimized for 10,000 steps using a conjugate gradient and line search
 algorithm native to NAMD. To improve simulation stability, the system was
 initially equilibrated using an NVT-ensemble with harmonic restraints
 placed on protein and lipid heavy atoms. The harmonic restraints were then
 incrementally relaxed over a period of 675 ps according to established
 CHARMM-GUI protocols (Lee et al., 2016). The system was then simulated at
 a constant pressure, utilizing the Langevin piston method to maintain 1
 atm at 303.15 K, from anywhere between 617 to 1176 ns depending on the
 starting conformation. Simulations were performed using periodic boundary
 conditions and a time step of 3.0 fs with all bonds to hydrogens being
 constrained. Large integration timesteps were enabled by employing
 hydrogen mass repartitioning (Hopkins, Le Grand, Walker, &
 Roitberg, 2015). Long-range electrostatic interactions were calculated
 using the particle mesh Ewald (PME) method with nonbonded interactions
 being cut off at 12 Å. Each simulation was performed in duplicate
 resulting in approximately 2 µs of total sampling for each system.
 Simulations are summarized in Supplementary Table 11.
 Code to generate the water occupancy data and analyze the
 simulations is located on GitHub: https://github.com/samberry19/nramp-md. More info on DraNramp can be found in our preprint: https://www.biorxiv.org/content/10.1101/2022.09.08.507188v1.
description: 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.