Structural basis of metal recognition by Tn21 MerR
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Tn21 MerR controls transcription of the mercury resistance (mer) operon located on transposon Tn21 from Shigella flexneri IncFII plasmid R100. Binding to the operator merO, MerR represses the transcription of merTPCAD in the absence of mercuric ions (Hg(II)) and activates transcription in the presence of Hg(II). MerR is ultrasensitive only to Hg(II), although other metals like Cd(II) and Zn(II) also induced MerR-mediated transcription from the mer promoter. The structural basis of MerR’s specificity for Hg(II) was poorly understood. In work described here, a polypeptide, MBD, was engineered by fusing two copies of the Hg(II)-binding domain of MerR in tandem. MBD retains MerR's ability to bind Hg(II) and establishes a very similar coordination environment for Hg(II). Both MerR and MBD can bind not only the natural inducer Hg(II), but also other thiophilic metal ions both in vivo and in vitro. Thus, MerR's high specificity for Hg(II) must reside at some step other than simply metal binding, and likely involves the whole protein and merO. Previous studies showed that when MerR is bound to merO the latter was distorted at the dyadic center by Hg(II). This dyad distortion was not observed with other metals, suggesting that DNA-bound MerR might still bind other metals but not have the same allosteric change as Hg(II) induces. Equilibrium dialysis revealed that DNA-bound MerR’s affinity for Hg(II) is lower than that of free MerR, and higher than free MerR for Cd(II) and Zn(II). 2-fluorotyrosine (2FY) was used as a sensitive probe to reveal the location of allosteric changes that MerR makes in distinguishing its natural inducer, 19Hg(II), from chemically similar metal ions Cd(II) and Zn(II). F NMR data of 2FY-MerR showed Y27, Y40, and Y46 of MerR occupy distinct environments and experience distinct allosteric changes in response to Hg(II), Cd(II) and Zn(II). DNA binding caused dramatic changes in the residues of MerR. Y27F and Y46F mutants demonstrated DNA binding deficiency, suggesting these two conserved tyrosines might be important links in DNA binding. Furthermore, two Cd(II)-responsive mutants K99T and M106V demonstrated unique conformational changes at DNA-binding domain, a possible direct reason for their losing specificity for Hg(II).