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.