THE INTERACTION BETWEEN THE METAL BINDING LOOP AND THE BACKBONE DETERMINES METAL-DIRECTED ACTIVATION OF MerR METALLOREGULATORS

MENDOZA JI; CHECA SK

Congreso; SAIB-SAMIGE Joint Meeting 2020 on line; 2020

Sociedad Argentina de Investigación Bioquímica y Biología Molecular y Sociedad Argentina de Microbiología General

Transcriptional regulation is the main cellular mechanism to control homeostasis and resistance to metal ions. Our group focuses on the MerR family of bacterial metalloregulators which are dimeric proteins that activate transcription of specific transporters or detoxification enzymes to eliminate surplus or toxic metal ions. The ability of these sensors to discriminate between metal ions is essential to achieve a proper response to a specific stress, and depends mainly on specific ligands such as cysteine or histidine residues sheltered at the metal coordination environment. Based on these key residues, two groups with similar structure can be clearly distinguished depending on the charge (+1 or +2) of the metal ion they sense. Two cysteine ligands define a loop in all MerR sensors, the metal-binding loop (MBL), that always has 7 residues in +1 metal sensors, but varies between 8 to 9 residues in those responding to +2 ions. Not only the length but also the identity of the residues within the loop varies between these regulators. While most of these sensors have low level of selectivity, as the ancestral CueR or ZntR sensors that respond to different +1 or +2 ions, respectively, some evolved to achieve preferential recognition to one specific metal ion, such as the Au(I) sensor GolS or the Hg(II) sensor MerR. Previously, we demonstrated that the MBL determines the selectivity of GolS for Au(I) ions. Also, we obtained a GolS derivative responsive to Hg(II) that carries the MBL of the Tn-501 MerR sensor and a third cysteine ligand at position 77. Here, we analyzed the contribution of the MBL to the evolution of monovalent and divalent metal sensors, and its role in the transduction of the detected signal to activate transcription. We applied site-directed mutagenesis and domain swapping generating a set of GolS, CueR and ZntR variants with modifications in both size and identity of the residues composing the MBL. The functionality of the mutant sensors was investigated by assessing activation of specific reporter genes followed by in silico modelling. Some of the variants modified the pattern of detected metal ions to privilege recognition of certain ions over others, including native inducers of the original sensor. However, this cannot be predicted because it not only depends on the size and sequence of the MBL, but also on the sensor backbone, particularly residues located in or close to the metal coordination environment. Altogether, these results provide the scientific rationale for fine tuning these biological sensors to achieve new, ligand-specific, sensing capabilities.