Two metal binding domains in the transcriptional regulator CRR1
Frederik Sommer1,2, Janette Kropat2, Davin Malasarn2, Nicholas E. Grossoehme3, Xiaohua Chen4, David P. Giedroc3, and Sabeeha Merchant2
1) MPIMP, Am Muehlenberg 1, 14476 Potsdam, Germany
2) Dept. Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095, USA
3) Dept. Chemistry, Indiana University, Bloomington, IN 47405, USA
4) Dept. Biochemistry Biophysics, Texas A&M, College Station, TX 77843, USA
CRR1 (Copper Response Regulator 1) in Chlamydomonas reinhardtii is essential to expresses genes like CYC6, CRD1 or CPX1 in response to copper deprivation. Thereby, copper containing proteins are replaced, anabolic pathways are changed and copper is relocated. CRR1 contains a plant specific DNA and Zn binding SBP domain with a new zinc finger fold, which can bind to copper response elements in the promoters of its target genes in vitro and activate their expression in vivo. When Chlamydomonas cells are treated with nickel or cobalt or grown anaerobically the same gene expression response can be observed in a CRR1-dependent manner. Remarkably, mercuric ions but not silver ions can mimic copper in turning off the copper deficiency response, which distinguishes the sensor from the mechanism operating in yeast and fungi. Expression of reporter constructs in mustard seedlings support nuclear localization of CRR1. When we tested two non Zn binding His residues of the SBP domain by point mutation, we found one of them to be crucial for DNA binding in in vitro using recombinant SBP domain. When the mutations were introduced into Chlamydomonas in vivo, the results were recapitulated in vivo. Additionally we could block DNA binding by the SBP domain in vitro by Cu(II) or Hg(II) additions while Ni(II) or Co(II) had no effect. Cu(I) also did not abolish SBP-DNA complexes in vitro although Cu(I) is able to replace Zn ions within the SBP domain without changing its conformation. Chlamydomonas mutants with a deletion of a C-terminal Cys rich domain in CRR1 were still responsive to Cu deficiency and Hg addition, whereas they did not respond any more to conditions of Ni addition or anaerobiosis. Taken together, this indicates separate responding sites for Cu and Hg vs. Ni and oxygen in CRR1.
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