Nickel-specific response in the transcriptional regulator, Escherichia coli NikR.

Studies of the transcriptional repression of the Ni-specific permease encoded by the Pnik 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 chromatography-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.