METAL ION-INTERACTION IN SYNTHETIC BROAD-SPECTRUM SENSORS DERIVED FROM THE Cu-RESPONSIVE CueR REGULATOR

LESCANO J; MENDOZA JI; SONCINI FC; CHECA SK

VIRTUAL

Congreso; LVII SAIB Meeting - XVI SAMIGE Meeting; 2021

SOCIEDAD ARGENTINA DE BIOQUIMICA Y BIOLOGIA MOLECULAR Y SOCIEDAD ARGENTINA DE MICROBIOLOGIA GENERAL

The Cu/Au/Ag CueR cytoplasmic sensor, a member of the MerR family of transcriptional regulators, is the main contributor to Cu homeostasis in Gram-negative bacteria. This biological sensor forms a dimer with two symmetrical metal binding sites formed by residues from both monomers. Cu(I), Ag(I) or Au(I) are coordinated in a linear array by two conserved cysteine residues (C112 and C120) that define the metal-binding loop (MBL). Within the metal coordination environment there is also a key serine residue (S77) from the other monomer that restricts the access of +2 ions to the metal binding site. S77 replacement for cysteine, the residue found in a similar position in all MerR members responding to +2 ions, allows the mutant CueR77 sensor from Salmonella to expand the spectrum of inducer metals to include Hg(II), Zn(II), Pb(II), Cd(II) or Co(II) ions. To understand the molecular bases directing metal recognition in this non-selective sensor, we introduced the same substitution (S77C) in the structurally characterized Escherichia coli (EC) CueR ortholog and evaluated its ability to interact with different divalent metals, both in vivo and in vitro comparing with the parental sensor. EC-CueR77 binds up to two equivalents of Hg(II), Cd(II) or Co(II) per dimer and all these metal ions are almost equally effective in switching the conformation of the regulator to its active form to induce transcription of its target genes. By contrast, the wild-type sensor only binds Hg(II), although with less affinity, and was unable to acquire the active conformation in these conditions. In addition, we generated a CueR77 derivative carrying the MBL of the Bacillus megaterium MerR mercury sensor (CueR77-LRB). This non-selective variant resulted fully insensitive to Cu(I) and poorly respond to Au(I) or Zn(II), but retained almost intact its ability to detect Pb(II) or Cd(II). Using competition assays with a specific Cu(I) chelator, we observed a significant decrease in its affinity for Cu compared with the parental sensor. Our results suggest that both the S77 residue and the native MBL region of CueR were coordinately selected during evolution of this biological sensor to avoid the interaction with +2 ions such as Zn(II) and guarantee a proper control of Cu homeostasis in bacteria.