title: Nickel binding to NikA: an additional binding site reconciles spectroscopy, calorimetry and crystallography.
abstract: Intracellular nickel is required by Escherichia coli as a cofactor for a number of enzymes and is necessary for anaerobic respiration. However, high concentrations of nickel are toxic, so both import and export systems have evolved to control the cellular level of the metal. The nik operon in E. coli encodes a nickel-uptake system that includes the periplasmic nickel-binding protein NikA. The crystal structures of wild-type NikA both bound to nickel and in the apo form have been solved previously. The liganded structure appeared to show an unusual interaction between the nickel and the protein in which no direct bonds are formed. The highly unusual nickel coordination suggested by the crystal structure contrasted strongly with earlier X-ray spectroscopic studies. The known nickel-binding site has been probed by extensive mutagenesis and isothermal titration calorimetry and it has been found that even large numbers of disruptive mutations appear to have little effect on the nickel affinity. The crystal structure of a binding-site mutant with nickel bound has been solved and it is found that nickel is bound to two histidine residues at a position distant from the previously characterized binding site. This novel site immediately resolves the conflict between the crystal structures and other biophysical analyses. The physiological relevance of the two binding sites is discussed.
authors.affiliation: Graduate School of Integrated Science, Yokohama City University, Tsurumi, Suehiro 1-7-29, Yokohama 230-0045, Japan.
authors.forename: Christine
authors.initials: C
authors.lastname: Addy
authors.forename: Masato
authors.initials: M
authors.lastname: Ohara
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Kawai
authors.forename: Akinori
authors.initials: A
authors.lastname: Kidera
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Ikeguchi
authors.forename: Sotaro
authors.initials: S
authors.lastname: Fuchigami
authors.forename: Masanori
authors.initials: M
authors.lastname: Osawa
authors.forename: Ichio
authors.initials: I
authors.lastname: Shimada
authors.forename: Sam-Yong
authors.initials: SY
authors.lastname: Park
authors.forename: Jeremy R H
authors.initials: JR
authors.lastname: Tame
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Heddle
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002151:Calorimetry; D003461:Crystallography; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D009532:Nickel / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013057:Spectrum Analysis
chemical_list: D018528:ATP-Binding Cassette Transporters; D029968:Escherichia coli Proteins; C089849:nikA protein, E coli; D009532:Nickel