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Metal specificity in nickel responsive transcriptional regulators

by Leitch, Sharon Virginia

Abstract (Summary)
Nickel is a required nutrient for bacteria. In E. coli, the regulation of nickel levels is achieved by NikR and RcnR, two nickel-responsive transcriptional regulatory proteins. Work herein illustrates connections between metal-site structure and protein function, proposing a mechanism for nickel-specific response. The high-affinity site of NikR is substituted with Co(II), Ni(II), Cu(II), Cu(I), and Zn(II) and the coordination environments characterized with XAS. The cognate nickel binds in a four-coordinate planar His3 Cys1 coordination environment. All other metals exhibit significant differences in coordination environment. Cu(II) and Zn(II) bind with the same ligands as Ni(II), however Cu(II) is distorted and Zn(II) is tetrahedral. Cu(I) is three coordinate, with a loss of an N/O-donor. Co(II) differs most compared to Ni(II). It is octahedral and is the only metal to lose the Cys S-donor. Concurrent results showed that different metals elicit different protein dynamics and function. These results implicate coordination geometry and ligand selection as a mechanism for metal-ion selectivity. Further structural studies of NikR are carried out on the low-affinity nickel site. The low-affinity site is six-coordinate and solvent accessible. RcnR is a recently characterized nickel-responsive regulatory protein. Herein, structure-function studies are used to characterize the metal binding sites. Ni-RcnR and Co-RcnR share many of the same ligands, but have distinct coordination spheres. Both metals have pseudooctaheral geometries, with a nickel coordination sphere of (NH2 )NHis3 NHis64 SCys35 (N/O) 2 and a cobalt coordination sphere of (NH2 )NHis3 N His60 NHis64 SCys35 (N/O). The possible role in metal recognition of the invariant cysteine ligand is examined for both NikR and RcnR. In NikR, the absence of C95 drastically reduces the binding affinity for nickel. In RcnR, C35 is required for cobalt function, but not nickel function, implicating it as a structural residue in Ni-RcnR and a functional residue, perhaps as an electronic sensor and point of allosteric communication, in Co-RcnR. The data herein supports a two part mechanism, cognate metal recognition through coordination environment elicits an allosteric response in the regulator, which alters DNA binding affinity.
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School:University of Massachusetts Amherst

School Location:USA - Massachusetts

Source Type:Master's Thesis

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Date of Publication:01/01/2008

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