Nickel-Specific Response in the Transcriptional Regulator, Escherichia coli NikR

摘要

Studies of the transcriptional repression of the Ni-specific permease encoded by the P_(nik) operon by Escherichia coli NikR using a LacZ reporter assay establish that the NikR response is specific to nickel in vivo. Toward understanding this metal ion-specific response, X-ray absorption spectroscopy (XAS) analysis of various M-NikR complexes (M = Co(Ⅱ), Ni(Ⅱ), Cu(Ⅱ), Cu(Ⅰ), and Zn(Ⅱ)) was used to show that each high-affinity binding site metal adopts a unique structure, with Ni(Ⅱ) and Cu(Ⅱ) being the only two metal ions to feature planar four-coordinate complexes. The results are consistent with an allosteric mechanism whereby the geometry and ligand selection of the metal present in the high-affinity site induce a unique conformation in NikR that subsequently influences DNA binding. The influence of the high-affinity metal on protein structure was examined using hydrogen/deuterium (H/D) exchange detected by liquid chromatog-raphy-electrospray ionization mass spectrometry (LC-ESI-MS). Each NikR complex gives rise to differing amounts of H/D exchange; Zn(Ⅱ)- and Co(Ⅱ)-NikR are most like apo-NikR, while the exchange time course is substantially different for Ni(Ⅱ) and to a lesser extent for Cu(Ⅱ). In addition to the high-affinity metal binding site, E. coli NikR has a low-affinity metal-binding site that affects DNA binding affinity. We have characterized this low-affinity site using XAS in heterobimetallic complexes of NikR. When Cu(Ⅱ) occupies the high-affinity site and Ni(Ⅱ) occupies the low-affinity site, the Ni K-edge XAS spectra show that the Ni site is composed of six N/O-donors. A similar low-affinity site structure is found for the NikR complex when Co(Ⅱ) occupies the low-affinity site and Ni(Ⅱ) occupies the high-affinity site, except that one of the Co(Ⅱ) ligands is a chloride derived from the buffer.