Nickel-Specific Response in the Transcriptional Regulator,EscherichiacoliNikR

Abstract

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.

Nickel Ions Inhibit Histone Demethylase JMJD1A and DNA Repair Enzyme ABH2 by Replacing the Ferrous Iron in the Catalytic Centers

“…X-ray Absorption Spectroscopy-XAS 3 data collection and analysis were performed as described previously (40). ABH2 samples (with a metal content of ϳ0.3 mM by inductively coupled plasma optical emission spectrometers) in 50 mM HEPES, pH 7.8, and 100 mM NaCl were syringed into polycarbonate sample holders with kapton windows and then frozen in liquid nitrogen.…”

“…Models involving all possible combinations of nitrogen and sulfur donors were addressed for each coordination number (see supplemental material). The number of histidine imidazole ligands involved in the coordination sphere was estimated by multiple-scattering analysis, as described previously (40). To compare different models to the same data set, ifeffit uses three goodness of fit parameters, 2 (Equation 1), reduced 2 , and R (Equation 2), where N idp ϭ number of independent data points; N⑀ 2 ϭ number of uncertainties to minimize; Re(fi) ϭ real part of the EXAFS fitting function; Im(fi) ϭ imaginary part of the EXAFS fitting function.…”

Uropathogenic enterobacteria use the yersiniabactin metallophore system to acquire nickel

“…E. coli acquire nutritional nickel ions via the conserved NikABCDE permease. Transcriptional upregulation of this system during UTI (9) is indicative of nickel deficiency during infection (10)(11)(12). This is consistent with nickel scarcity in human hosts, where urinary content (~0.04 µmoles per liter) is lower than that of iron (~0.1 µmoles per liter) (13, 14).…”

Geobacter uraniireducensNikR Displays a DNA Binding Mode Distinct from Other Members of the NikR Family

“…Nickel binding to the MBD activates NikR to bind to specific DNA sequences and regulate gene expression (1,6,8,13,23,27). Escherichia coli NikR (EcNikR) was the first member of this large family of transcription factors to be studied (11,18), and its function is relatively well understood (11,13,18,27,36,46).…”

“…IPTG (isopropyl-␤-D-thiogalactopyranoside) was not added to the growth medium. LacZ assays were performed as described previously (11,13,18,27,36,46). in the supplemental material) revealed a high degree of variability in ␤-sheet and arm sequence, as well as in arm length.…”