Conformational and thermodynamic hallmarks of DNA operator site specificity in the copper sensitive operon repressor from Streptomyces lividans

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.

Cu(I)-mediated Allosteric Switching in a Copper-sensing Operon Repressor (CsoR)

“…4D), which are identical for all Cu(I)-specific CsoRs. None of these clade-specific residues are found, for example, in M. tuberculosis CsoR, but all must be superimposed on conserved residues probably important for DNA binding (26). The same is more generally true for Ni(II)/Co(II)-specific RcnR (group IIa; Fig.…”

“…If DNA binds across the one face of the tetramer and over the "hole" (see Fig. 4C), as recent studies taken collectively indicate (26,27), such a change in quaternary structure would probably drive disassembly of the 2:1 CsoR-DNA complex (Fig. 5A).…”

“…Two copper-free crystal structures subsequently reported for T. thermophilus and S. lividans CsoRs adopt the same dimer-of-dimers architecture (18,20). ApoCsoR binds specifically to its operator DNA in a 2:1 tetramer/ operator binding stoichiometry (16,26) but lacks a canonical DNA binding domain; as a result, precisely how CsoR interacts with its pseudo-2-fold symmetric DNA operator remains unclear. A recent study presents a plausible model of how two CsoR tetramers are oriented on a single DNA operator (26), whereas a mass spectrometry-based method used to probe differential lysine reactivity on B. subtilis CsoR provided some insights into the conformations in distinct allosteric states (27).…”

“…ApoCsoR binds specifically to its operator DNA in a 2:1 tetramer/ operator binding stoichiometry (16,26) but lacks a canonical DNA binding domain; as a result, precisely how CsoR interacts with its pseudo-2-fold symmetric DNA operator remains unclear. A recent study presents a plausible model of how two CsoR tetramers are oriented on a single DNA operator (26), whereas a mass spectrometry-based method used to probe differential lysine reactivity on B. subtilis CsoR provided some insights into the conformations in distinct allosteric states (27). However, how Cu(I) binding drives negative regulation of DNA binding is not known for any CsoR or, more generally, any CsoR/RcnR protein.…”

Mycobacterium tuberculosis and Copper: A Newly Appreciated Defense against an Old Foe?

“…Interestingly, RicR is more similar to CsoR orthologues in other bacteria than M. tuberculosis CsoR. The crystal structure of the RicR orthologue in Streptomyces lividans (CsoR) revealed a dimer of tetramers configuration (16 3 Furthermore, deletion of mmcO in M. tuberculosis results in a Cu-sensitive phenotype that is observed using an agar plate-based assay, but not a liquid-based assay. This result differs from the more robust Cu-sensitive phenotype observed with a mymT mutant that has phenotypes both in liquid and on solid medium (12,18,22).…”