title: Ferric uptake regulator protein: binding free energy calculations and per-residue free energy decomposition.
abstract: Iron homeostasis is, in many bacterial species, mediated by the ferric uptake regulator (Fur). A regulatory site able to bind iron to activate Fur for DNA binding has been described, and a structural zinc site essential for the dimerization has also been proposed. They have been localized and named site 1 and site 2, respectively, from the crystal structure of a zinc-substituted Pseudomonas aeruginosa Fur (PA-Fur). Notwithstanding the studies on Fur proteins from various species, both the precise site of iron binding and the effect on DNA binding affinity are still controversial. These issues were investigated here by molecular dynamics simulations and free energy calculations. Simulations were performed for eight molecular systems represented by the three forms of Fur, that is, apo Fur, metal-substituted Fur, and Fur complexed with DNA. Because of the lack of a Fur-DNA complex crystal structure, the recently published model based on mass spectrometry experiments on Escherichia coli Fur (EC-Fur), and the crystal structure of PA-Fur, was used, after adjustment to adopt a symmetric conformation. The simulation results suggest that the formerly proposed site 2 is, in fact, the regulatory iron-sensing site. The calculations also predict that Fe(2+) at site 2 is hexacoordinated having an octahedral environment with only nitrogen and oxygen atoms, which is in accordance with previous spectroscopic characterizations. Energy decomposition pinpoints H87 as an additional amino acid that defines the regulatory metal site. Finally, free energy decomposition analysis reveals a number of amino acids potentially important in dimerization and in DNA binding.
authors.affiliation: Department of Molecular Biotechnology, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, N-9037, Tromsø, Norway.
authors.forename: Rafi
authors.initials: R
authors.lastname: Ahmad
authors.forename: Bjørn Olav
authors.initials: BO
authors.lastname: Brandsdal
authors.forename: Isabelle
authors.initials: I
authors.lastname: Michaud-Soret
authors.forename: Nils-Peder
authors.initials: NP
authors.lastname: Willassen
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D004247:DNA; D007501:Iron