Ludovic Pecqueur
Role of the zinc and the cysteines in the dimerization of the Ferric Uptake Regulator (FUR) from E. coli: A structural approach using NMR
Published on 14 December 2005
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Thesis presented December 14, 2005
Abstract:
The FUR protein (Ferric Uptake Regulator) is a global regulator ubiquitous in Gram-negative bacteria. Binding of Fe2+in vivo activates the FUR protein for DNA binding. Fe-FUR binds to the promotor of FUR regulated genes, causing their repression. This work consists of an NMR structural study of the non-activated dimer of FUR (2*17 kDa) which contains one zinc ion per monomer. The FUR monomer was also studied and does not contain the zinc ion. Reduction of the cysteines, and binding of zinc to the protein, leads to dimerization. Circular dichroism and NMR showed that folding of the C-terminal domain of the monomer (dimerization domain) occurs upon dimerization. The secondary structure observed in the monomer and the dimer are similar, except for the first residues. In the monomer, these are organized as a helix while they are unfolded in the non-activated dimer. As this first helix is observed in the activated dimer of
P. aeruginosa, we propose it plays a role in the regulation mechanism. A truncated protein named FUR1-82 was constructed, purified, and crystallized. Its structure is superimposable onto the N-terminal domain structure of
P. aeruginosa FUR and the
1H-15N-HSQC spectra of FUR1-82 and FUR monomer are nearly identical. Binding of the monomer and the dimer to the DNA consensus sequence study, using fluorescence anisotropy, showed that both forms are able to bind DNA specifically, but with different affinities. The monomer has a 5 fold decreased affinity compared to the dimer in presence of excess metal. Overall, the results obtained allowed us to propose a dimerization mechanism of
E. coli FUR and an activation mechanism with a role for the N-terminal alpha helix.
Keywords:
Dimerization, FUR, Ferric Uptake Regulator, cysteines, zinc, NMR, circular dichroism, fluorescence anisotropy, crystallography
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