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April 9, 2003 Coordination chemistry of nickel uptake regulation in Escherichia coliP.E. Carrington1, M.J. Maroney1, R.T. Sauer2, P.T. Chivers2,3, and F. Al-Mjeni1,4 Scientists from the University of Massachusetts in Amherst and the Massachusetts Institute of Technology in Cambridge have determined the structure of the high affinity nickel-binding site in NikR, a protein that regulates the uptake of nickel by the gut bacterium Escherichia coli. The scientists show that, in the NikR protein, nickel is bound in a novel four-coordinate planar site consisting of two histidines, one additional oxygen- or nitrogen-donor ligand, and one sulfur-donor (cysteine) ligand. The researchers also noticed that when NikR binds to DNA, the nickel-binding site becomes six-coordinate with ligands made of oxygen and nitrogen donors, but lacking cysteine.
This protein is produced via transcription of the nik operon (chromosomal functional unit acting like a gene) in response to low oxygen level. When nickel concentration is high enough, the transcription process is repressed by NikR, a protein that appears to regulate nickel uptake in a number of bacteria and archaea. NikR contains two distinct binding domains: an amino terminal, DNA-binding site and a carboxyl terminal high-affinity nickel-binding site. By using x-ray absorption spectroscopy (XAS) at beamline X9B at the NSLS, we have characterized the structure of the high-affinity nickel-binding site and have shown that the nickel-binding site is sensitive to the DNA-bound state of NikR. Analysis of x-ray absorption near-edge structure (XANES) revealed
that the nickel site is four-coordinate and planar, because it
exhibits a 1s → 3d electronic transition near 8332 electronvolts (eV)
and a distinct maximum assigned to a 1s → 4pz transition near 8338 eV.
The nickel-binding domain of NikR contains a number of conserved amino acids that are potential nickel ligands. The structure that emerges from the combined XAS and mutagenesis of four of these amino acids is consistent with the planar four-coordinate N2OS-donor site shown in figure 1. Since nickel is the only important biological metal ion that commonly adopts a square planar geometry, the results provide a structural basis for the specificity of NikR toward nickel ions. Large structural changes were observed when NikR was bound to operator DNA (figure 2). The XANES spectra of the NikR-DNA complexes (left) exhibit somewhat larger, but still small, peaks associated with 1s → 3d electronic transitions near 8332 eV, while the peak assigned to the 1s → 4pz transition near 8338 eV is absent, indicating the presence of a six-coordinate nickel site. EXAFS analysis of the DNA complexes (right) shows that the best fits are obtained for six nitrogen and oxygen donors, including at least two histidine ligands, consistent with the XANES analysis.
NikR appears to be using the coordination chemistry characteristic of nickel not only to selectively bind nickel, but also to change the interaction between nickel and the protein. The change in nickel-coordination geometry has several potential consequences for NikR function, such as its role for NikR in buffering intracellular nickel at very low levels and controlling DNA transcription at higher concentrations of intracellular nickel. Although the functional consequences of the change in nickel coordination remain to be elucidated, NikR provides a unique example of the role of metal ions in regulating DNA transcription. BEAMLINE FUNDING PUBLICATION FOR MORE INFORMATION |
The National Synchrotron Light Source at Brookhaven National Laboratory in New York, is a national user research
facility funded by the U.S. Department of Energy's Office of Basic Energy Science. The NSLS operates two electron
storage rings: an X-Ray ring and a Vacuum UltraViolet (VUV) ring which provide intense light spanning the electromagnetic
spectrum from the infrared through x-rays. Each year over 2300 scientists from universities, industries and government
labs perform research at the NSLS.
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Microorganisms have developed high-affinity uptake systems to
acquire metals, such as iron, copper, and zinc, from the environment.
In the gut bacterium Escherichia coli, nickel plays a critical role
in anaerobic metabolism, but it is usually present at low
concentrations in the environment, so E. coli synthesizes a protein
called NikABCDE, which acquires nickel from the environment and
actively transports it into the cell. (The letters A to E label five
proteins that assemble to form NikABCDE.) 
This result was confirmed by the analysis of extended x-ray
absorption fine structure (EXAFS), which revealed that the distances
between nickel and other atoms are typical of planar four-coordinate
nickel complexes, and also provided information about the ligands
involved. 