Abstract
Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resistance genes and enable the organism to preclude metal toxicity. The copper sensitive operon repressor (CsoR) family is widely distributed in bacteria and controls the expression of copper efflux systems. CsoR operator sites consist of G-tract containing pseudopalindromes of which the mechanism of operator binding is poorly understood. Here, we use a structurally characterized CsoR from Streptomyces lividans (CsoRSl) together with three specific operator targets to reveal the salient features pertaining to the mechanism of DNA binding. We reveal that CsoRSl binds to its operator site through a 2-fold axis of symmetry centred on a conserved 5′-TAC/GTA-3′ inverted repeat. Operator recognition is stringently dependent not only on electropositive residues but also on a conserved polar glutamine residue. Thermodynamic and circular dichroic signatures of the CsoRSl–DNA interaction suggest selectivity towards the A-DNA-like topology of the G-tracts at the operator site. Such properties are enhanced on protein binding thus enabling the symmetrical binding of two CsoRSl tetramers. Finally, differential binding modes may exist in operator sites having more than one 5′-TAC/GTA-3′ inverted repeat with implications in vivo for a mechanism of modular control.
Highlights
In bacteria, families of metal sensing transcriptional regulators, commonly referred to as metalloregulatory or metal sensor proteins, act to control the expression of genes that allow the organism to quickly adapt to chronic toxicity or deprivation of biologically essential metal ions [1,2,3,4]
We report that CsoR from Streptomyces lividans (CsoRSl) binding drives a ‘conformational switching’ in the operator DNA that is dependent on the inherent deformability brought about by the G/C-tracts within the operator site
Using the operator site with the longest consensus sequence, csoR-4375, the thermodynamics of binding to apo-CsoRSl were first investigated by isothermal titration calorimetry (ITC)
Summary
Families of metal sensing transcriptional regulators, commonly referred to as metalloregulatory or metal sensor proteins, act to control the expression of genes that allow the organism to quickly adapt to chronic toxicity or deprivation of biologically essential metal ions [1,2,3,4] These proteins are able to form specific metal ion coordination complexes, with metal affinities as high as femto- to zeptomolar for Cu(I) and some Zn(II) sensors [5,6], and can either inhibit or activate operator DNA binding or directly enhance transcriptional activation [2]. Only the copper sensitive operon repressor (CsoR) proteins from Mycobacterium tuberculosis [7], Thermus thermophilus [10] and more recently from Streptomyces lividans [11] have been determined and all in the absence of operator DNA These CsoR proteins exist in solution as tetrameric assemblies with each protomer consisting of three a-helices of varying lengths (Figure 1). One coordinating Cys and His residue are positioned towards the C-terminal end of the a2 helix, and the second Cys ligand is located at the N-terminal end of the a20 helix of a second protomer (Figure 1)
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