Integrating the Molecular Machines of Mercury Detoxification into Host Cell Biology

Abstract

Metalloregulators of the MerR family activate transcription upon metal binding by underwinding the operator-promoter DNA to permit open complex formation by pre-bound RNA polymerase. Historically, MerR's allostery has been monitored only indirectly via nuclease sensitivity or by fluorescent nucleotide probes and was very specific for Hg(II), although purified MerR binds several thiophilic metals. To observe directly MerR's ligand-induced behavior we made 2-fluorotyrosine-substituted MerR and found similar, minor changes in 19 F chemical shifts of tyrosine residues in the free protein exposed to Hg(II), Cd(II) or Zn(II). However, DNA binding elicits large chemical shift changes in MerR's tyrosine residues and in DNAbound MerR Hg(II) provokes changes very distinct from those of Cd(II) or Zn(II). These chemical shift changes and other biophysical and phenotypic properties of wild-type MerR and relevant mutants reveal elements of an allosteric network that enables the coordination state of the metal binding site to direct metal-specific movements in the distant DNA binding site and the DNA-bound state also to affect the metal binding domain. IntroductionMetal homeostasis, the timely and specific movement of beneficial metal ions from outside the cell into their appropriate intracellular niches as structural or catalytic cofactors, is an essential process in all organisms, effected by membrane transporters and cytoplasmic metallochaperones controlled by metalloregulators.1 The molecular mechanisms enabling such proteins to make distinct responses to chemically similar metal ligands are just beginning to be elucidated, but recent work underscores the need for appropriately arranged protein ligands to support effective metal occupancy. [2][3][4] In addition to genes for homeostasis of beneficial metals, bacteria frequently have genes that afford survival of exposure to toxic metals; the transcriptional regulators of such metal resistance systems are often homologs of those managing homeostasis of essential metals. 5 One of the largest such metalloregulator families is named for the transcriptional repressor-activator of the bacterial mercury resistance (mer) operon, MerR, and includes CueR, ZntR, CoaR, PbrR, and SoxR that control bacterial resistance and/or homeostasis to Hg(II), Cu(I/II), Zn(II), Co(II) and Pb(II/III) and the Fe(II/III)-mediatedresponse to superoxide, respectively. There is a second distinct branch within the MerR family comprised of aromatic-compound-responsive regulators such as BmrR and Mta that control multidrug efflux pumps 5 ( Figure 1). In promoters regulated by MerR-family members, a dyadic operator lies within an overlong spacer between good consensus −10 and −35 recognition sites for the major cellular RNA polymerase. MerR bends its operator DNA (MerOP) 6,7 and surprisingly represses transcription of the structural genes merTPCAD by capturing RNA polymerase (RNAP) in a stable but inactive pre-initiation complex. [8][9][10][11] When Hg(II) binds to DNA-bound MerR, the protein underwinds the...