Protease digestion analysis of Escherichia coli NikR: evidence for conformational stabilization with Ni(II).
The Escherichia coli NikR is a 15-kDa protein that negatively regulates transcription of the nikABCDE operon that encodes for an ATP-dependent Ni(II) permease. Thermal and chemical denaturation studies with NikR previously demonstrated that Ni(II)-NikR is more stable than the protein bound to other metals such as Cu(II), Co(II) and Zn(II). To determine if Ni(II) induces a unique conformational change in NikR, digestion experiments with selected proteases were performed in the presence of the above metals. Both denaturing-polyacrylamide gel electrophoresis and reversed-phase HPLC revealed fragmentation patterns in the presence of stoichiometric nickel that were distinct from the cleavage of apo-NikR. Digestion of Cu(II)-NikR produced fragmentation that was similar, although less dramatic, to that produced with Ni(II)-NikR, whereas the Zn(II)- and Co(II)-bound proteins were digested in a similar manner as apo-NikR. Digestion fragments were collected, identified by MALDI-MS, and then mapped onto the available crystal structure of NikR. Although the specificity of the proteases utilized differed, the data suggest that Ni(II) has a selective allosteric effect and that upon metal binding the NikR metal-binding pocket is oriented or protected in such a way as to present itself for digestion in a unique conformation. This data sheds light on the Ni(II)-selective conformational changes that allow NikR to bind DNA optimally and repress transcription of the nik operon.
Dias
Alistair V
AV
Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, M5S 3H6, Canada.
Zamble
Deborah B
DB
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003300:Copper; D003461:Crystallography; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009532:Nickel / Q000737:chemistry*; D010196:Pancreatic Elastase / Q000737:chemistry; D010449:Peptide Mapping; D011487:Protein Conformation; D011489:Protein Denaturation; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry
D029968:Escherichia coli Proteins; C402409:NikR protein, E coli; D012097:Repressor Proteins; D003300:Copper; D009532:Nickel; D010196:Pancreatic Elastase; D014357:Trypsin