The ubiquitous Nramp family of transport proteins, a member of the LeuT family, facilitates acquisition of metal ions, including the absorption of dietary iron in mammals. Nramp proteins have an essential role in host defense against pathogens. Thus, known Nramp mutations lead to impaired immune response. Furthermore, since Nramp is also crucial for iron uptake into the cytosol, its mutations cause anemia in humans and rodents. Here, tools developed at the Center will be applied to resolve (1) how Nramp selectively binds and transports essential transition metal ions (Fe2+, Co2+, Mn2+, and Zn2+) over toxic metals (Cd2+ and Pb2+) or the more abundant alkaline earth metals (Ca2+ and Mg2+), and (2) why Nramp mutants fail to achieve such selectivity for transition metals.

A problem that needs to be overcome is that only a low-resolution (4.5 Å) crystallographic structure capturing Nramp's metal-bound state is available, which does not resolve suciently the metal ion binding site. The xMDFF method developed at the Center (TRD3) will be employed to improve the atomic structure of Nramp from the low-resolution crystallographic data. The metal ion binding and transport of Nramp depends on the protonation state of its four binding-site residues (two ASP, one MET, and one HIS). A second difficulty is the determination of the binding-site protonation states and how changes in these states affect metal ion coordination. This difficulty will be addressed with constant-pH simulations of the metal-bound Nramp (TRD1). The third computational problem arises from the millisecond timescale of metal ion transport across Nramp, which is well beyond the reach of classical MD simulations. This difficulty will be overcome by employing a combination of MD, steered MD (SMD) and path sampling simulations together with alchemical free energy calculations, all of which were developed at the Center and implemented in NAMD (TRD1).

State of the work

The Center has a strong history of studying ion channels and transporters, having on the one hand contributed to discoveries of ion transport mechanisms, while on the other hand developed tools and resources to simulate the coupled dynamics of proteins with the transporting ions. Recently, employing xMDFF (TRD3) with low-resolution X-ray diffraction data (Gaudet) a detailed atomic model of Fe2+-bond Nramp has been determined. The MD and SMD simulations of this model with bound Fe2+ and Ca2+ ions reveal that Fe2+ interacts with the sulfur atom of a methionine residue, M226, via a soft acid-soft base interaction, thus binding to Nramp with twice as much strength as does Ca2+, which interacts only minimally with M226 via a hard acid-soft base interaction. Altogether, a mechanism explaining ion selectivity of divalent metal ion membrane transporters based upon differential M226 binding has been proposed.

Research plan

In the upcoming research, the metal ion transport across Nramp and a M226A Nramp mutant will be studied. First, constant-pH simulations (TRD1) will be employed with Fe2+-, Zn2+- and Ca2+-bound Nramp, to determine how changes in protonation state of the binding-site ASP and HIS residues alter the metal ion coordination. The most probable metal ion-coordination geometries will be identified for each one of the three ions. Second, to validate the accuracy of the predicted coordination geometries, metal-ion binding anities will be derived from these coordinations employing alchemical free energy calculations on NAMD (TRD1), and compared to those obtained experimentally from binding assays (Gaudet). Finally, in analogy to other transporters in the LeuT family, changes in the metal ion binding coordination with binding-site protonation states is expected to induce conformational transitions of the transporter; these transitions are known to yield intermediates characterizing the ion transport pathway across the membrane. Since constant- pH simulations only depict initial stages of the millisecond-scale event, the entire transition will be captured employing the path sampling features of NAMD (TRD1). The aforementioned three-step computations will then be repetaed with the M226A Nramp mutant to elucidate the role of M226 in transport. The computationally predicted intermediate-stabilizing residues will guide further mutagenesis, crystallographic and transport activity studies of Nramp (Gaudet).

Publications Database
  • Conserved methionine dictates substrate preference in Nramp-family divalent metal transporters. Aaron T. Bozzi, Lukas B. Bane, Wilhelm Weihofen, Anne McCabe, Abhishek Singharoy, Chris Chipot, Klaus Schulten, and Rachelle Gaudet. Proceedings of the National Academy of Sciences, USA, 113:10310-10315, 2016.
  • Atomic-level characterization of transport cycle thermodynamics in the glycerol-3-phosphate:phosphate transporter. Mahmoud Moradi, Giray Enkavi, and Emad Tajkhorshid. Nature Communications, 6:8393, 2015.
  • Computational recipe for efficient description of large-scale conformational changes in biomolecular systems. Mahmoud Moradi and Emad Tajkhorshid. Journal of Chemical Theory and Computation, 10:2866-2880, 2014.
  • Mechanistic picture for conformational transition of a membrane transporter at atomic resolution. Mahmoud Moradi and Emad Tajkhorshid. Proceedings of the National Academy of Sciences, USA, 110:18916-18921, 2013.
  • Calculation of the gating charge for the Kv1.2 voltage-activated potassium channel. Fatemeh Khalili-Araghi, Vishwanath Jogini, Vladimir Yarov-Yarovoy, Emad Tajkhorshid, Benoit Roux, and Klaus Schulten. Biophysical Journal, 98:2189-2198, 2010.
  • Molecular dynamics simulations of membrane channels and transporters. Fatemeh Khalili-Araghi, James Gumbart, Po-Chao Wen, Marcos Sotomayor, Emad Tajkhorshid, and Klaus Schulten. Current Opinion in Structural Biology, 19:128-137, 2009.
  • Dynamics of K+ ion conduction through Kv1.2. Fatemeh Khalili-Araghi, Emad Tajkhorshid, and Klaus Schulten. Biophysical Journal, 91:L72-L74, 2006.
  • Structural mechanism of plant aquaporin gating. S. Törnroth-Horsefield, Y. Wang, K. Hedfalk, U. Johanson, M. Karlsson, E. Tajkhorshid, R. Neutze, and P. Kjellbom. Nature, 439:688-694, 2006.
  • What makes an aquaporin a glycerol channel: A comparative study of AqpZ and GlpF. Yi Wang, Klaus Schulten, and Emad Tajkhorshid. Structure, 13:1107-1118, 2005.
  • Free energy calculation from steered molecular dynamics simulations using Jarzynski's equality. Sanghyun Park, Fatemeh Khalili-Araghi, Emad Tajkhorshid, and Klaus Schulten. Journal of Chemical Physics, 119:3559-3566, 2003.