Thomas V. O'Halloran
Board Member: 2010-2013
It is surprising how little is known about the cell biology of the transition metals. Living systems respond to heavy metal stress at several levels, depending whether the stress involves starvation or overload of an essential metal such as copper, or an excess of a nonessential metal such as mercury. Using methods from the fields of physical inorganic chemistry, molecular, and cell biology, we study the mechanisms of these processes. The initial reaction of many cells to heavy metal stress is activation of genes involved in detoxification or uptake. Our first goal is the characterization of the intracellular "metal sensing" apparatus, i.e., metalloregulatory proteins. These DNA binding proteins are metal responsive molecular switches that sense changes in metal concentration and adjust the expression of relevant genes.
Mechanistic and structural studies of one of the first characterized metalloregulatory proteins, MerR, are underway. MerR exerts both positive and negative control over bacterial mercury resistance operons, and it is a DNA-binding protein specific for the mer promoter. This small, 144-residue protein represses gene expression in the absence of mercury and apparently activates gene expression in the presence of mercury, using a novel DNA distortion mechanism. In this case, an allosteric change in protein conformation is induced by Hg(II) binding and then transmitted to the transcriptional machinery through a change in DNA conformation. Essentially, Hg-MerR changes the twist and bend of its DNA binding site and thereby makes the promoter a better substrate for the RNA polymerase. It is likely that some other stress responsive operons (such as SoxR) will use similar mechanisms. Other microbial metal receptors, including the virulence factor and iron sensor Fur and the copper-sensors PcoS/PcoR, are under study.
Metal responseive gene transcription plays an important role in eukaryotic heavy metal homeostasis systems. Our current eukaryotic targets include the isolation and characterization of proteins included in Zn and Cu-transport and utilization. Characterization of the proteins will reveal fundamental molecular aspects of heavy metal homeostasis and may provide insight into fatal metal based disorders in humans, such as Menke's syndrome and Wilson's disease. To further underand the cell biology of zinc, we are developing a series of fluorescent probes that can indicate changes in intracellular Zn(II) concentrations.
We are also investigating the chemistry of small iron complexes and nonheme iron proteins, which are thought to be involved in lipid peroxidation, heavy metal homeostasis, and dioxygen addition biological substrates. We have eludicated aspects of metallobiochemistry common to both toxic and essential metals and eventually plan to link our results at the molecular level to specific phenomena at the cellular level.
Investigation of this new class of metalloproteins enhances our understanding of several important phenomena. It reveals how metals can direct changes in the structure and function of nucleic acid binding proteins and provides insights into new mechansims of gene regulation. Finally, these studies provide molecular insights into the metabolism of essential metals and the effects of toxic metals on microbes and humans.