Specific amino acid residues in GolS metal-binding loop determines the metal selectivity

MARÍA M. IBÁÑEZ; SUSANA K. CHECA; FERNANDO C. SONCINI

Rosario

Congreso; V Congreso Argentino de Microbiología General; 2008

Sociedad Argentina de Microbiología General (SAMIGE)

Metal-dependent transcriptional regulatory systems are the major cellular mechanism that bacteria employ to control metal homeostasis and/or resistance to harmful non-essential transition elements. Salmonella has two transcriptional regulators of the MerR family that respond to monovalent metal ions: CueR, which is involved in copper homeostasis, and GolS, which is responsible for gold detoxification. In contrast to CueR, that has similar affinity for both cations, GolS is 52 times more sensitive to Au than to Cu. A detailed analysis of the C-terminal metal-binding loop of these proteins (between two conserved Cys: C112 and C120) shows some differences that could account for cation-specificity. Indeed, while CueR harbor a CPGDDSADC sequence, a CAGDALPDC sequence is present in GolS, highlighting the switch between P113 and P118, and the presence of a charged D residue in position 116 in CueR, that in GolS is an A.To investigate the role of this loop in metal discrimination we first generated chimeric GolS and CueR sensors in which their native metal-binding loop were replaced by the one present in CueR or GolS, respectively. We observed that the GolS chimeric protein (with the metal-binding loop of CueR) was able to activate the expression of the GolS-controlled gene golB, by the addition of Au or Cu ions in a similar manner than the induction of a typical CueR-regulated gene. Conversely, expression of the CueR-controlled gene copA was induced by the CueR chimeric protein (with the metal-binding loop of GolS) only by the addition of Au ions. In addition, we constructed by site-directed mutagenesis GolS mutant proteins in which the amino acids within the metal binding loop of the sensor that distinguish the GolS grove was replaced with those present in CueR-like regulators. The response of each mutant regulator to Au, Cu or Ag ions was analyzed by monitoring golB expression. While induction of golB was only achieved by Au in the wild type strain and in most GolS mutants, it was induced to a similar extent either by the addition of Au, Cu or Ag in a A113P mutant strain.Our results indicate that in GolS Au-selectivity is achieved by the amino acid residues encompassing the C112-C120 loop, and highlight the relevance of position 113 for the discrimination between the monovalent metal-ions from group IB of the periodic table.