INVESTIGADORES
CHECA Susana Karina
congresos y reuniones científicas
Título:
FUNCIONAL AND SPECTROSCOPIC CHARACTERIZATION OF A SYNTHETIC NONSELECTIVE METALLOSENSOR
Autor/es:
LESCANO J; SONCINI FC; CHECA SK
Reunión:
Congreso; SAIB-SAMIGE Joint Meeting 2020 on line; 2020
Institución organizadora:
Sociedad Argentina de Investigación Bioquímica y Biología Molecular y Sociedad Argentina de Microbiología General
Resumen:
CueR is a widely distributed member of MerR family of metal sensors and transcriptional regulators from Gram-negative bacteria that directs copper detoxification through activation of envelope-located factors. Besides intracellular Cu(I), CueR senses Ag(I) and Au(I) with similar zeptomolar affinity. These metal ions interact in a linear bi-coordinate geometry with the -S groups of C112 and C120, two cysteine residues conserved in all members of the family. In the functional CueR dimer, these cysteines and other residues from both monomers, including the key serine residue (S77), form two metal-binding sites from which the inducing signal is transduced to the parallel DNA-binding motifs to activate transcription of the target genes. We previously demonstrated that the S77 residue, conserved in all CueR-like sensors, is essential for preserving the in vivo selectivity for +1 metal ions exhibited by the Salmonella CueR and GolS sensors. Replacement of the S77 residue in either CueR or GolS for cysteine, the residue found in a similar position in all MerR members responding to Hg(II), Zn(II), Pb(II) or Cd(II), allows the mutant -S77C sensors to activate transcription of the target genes in response to their native +1 inducers, but also in the presence Hg(II). In fact, in a strain deleted in the major Zn(II)/Pb(II)/Cd(II) transporter, ZntA, the mutant sensors were also responsive to Pb(II), Cd(II) or Co(II) salts. To understand the molecular bases directing metal recognition in these non-selective sensors, we reproduced the S77C mutation in the Escherichia coli CueR orthologue (CueREC), for which structural data is available, and validated the response of CueREC-S77C sensor to +1 and +2 metal ions in vivo. Using spectroscopic analysis, we tested and compared the interaction of purified CueREC-S77C and the wild-type sensor with different divalent metals. We found that the mutant sensor binds up to two equivalent of Hg(II), Cd(II) or Co(II) using the additional cysteine ligand. These results confirm the in vivo observations and contribute to understand the key role of the residue at position 77 for metal-binding and discrimination in this family of metal sensor/regulators