The ars operon of the Escherichia coli plasmid R773 that confers arsenical and antimonial resistance is negatively regulated by the ArsR repressor. ArsR residues Cys-32 and Cys-34 were previously identified as involved in induction by arsenite and antimonite, suggesting coordination between As(III) and the two cysteine thiolates. However, in small molecule thiolate-As(III) complexes, arsenic is frequently three-coordinate. A site-directed mutagenic approach was employed in a search for a third arsenic ligand. ArsR proteins with C32G, C34G, and C32G/C34G substitutions were active repressors, but were not inducible in vivo. In vitro, the altered repressor-ars DNA complexes could not be dissociated by inducers. Alteration of Cys-37 and Ser-43, residues located in or near the putative helix-turn-helix DNA-binding region of the protein, had no effect on the inducibility of the operon. While these results indicated that neither the thiolate of Cys-37 nor the hydroxyl oxygen of Ser-43 is required for induction, they did not eliminate either atom as a potential arsenic ligand. Another approach involved reaction with an alternative inducer, phenylarsine oxide, which can form only two coordinations. Phenylarsine oxide was shown to be as effective as or more effective than arsenite or antimonite in induction in vivo. In vitro, the organic arsenical was more effective than either arsenite or antimonite in dissociating the repressor-promoter complex. Thus, two ArsR-arsenic bonds are sufficient for induction. The interaction of ArsR proteins with As(III) was examined using a phenylarsine oxide affinity resin. ArsR proteins containing any two of the three cysteine residues Cys-32, Cys-34, and Cys-37 bound to the resin. Alteration of any two of the three resulted in loss of binding. Arsenic x-ray absorption spectroscopy of ArsR treated stoichiometrically with arsenite confirmed the average arsenic coordination as AsS. These results suggest that all three cysteine thiolates are arsenic ligands, but binding to only two, the Cys-32 and Cys-34 thiolates, is required to produce the conformational change that results in release of the repressor from the DNA and induction.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				K. Yoshida, K. Inoue, Y. Takahashi, S. Ueda, K. Isoda, K. Yagi, and I. Maeda Novel Carotenoid-Based Biosensor for Simple Visual Detection of Arsenite: Characterization and Preliminary Evaluation for Environmental Application Appl. Envir. Microbiol., November 1, 2008; 74(21): 6730 - 6738. [Abstract] [Full Text] [PDF]

				J. Qin, H.-L. Fu, J. Ye, K. Z. Bencze, T. L. Stemmler, D. E. Rawlings, and B. P. Rosen Convergent Evolution of a New Arsenic Binding Site in the ArsR/SmtB Family of Metalloregulators J. Biol. Chem., November 23, 2007; 282(47): 34346 - 34355. [Abstract] [Full Text] [PDF]

				D. R. Campbell, K. E. Chapman, K. J. Waldron, S. Tottey, S. Kendall, G. Cavallaro, C. Andreini, J. Hinds, N. G. Stoker, N. J. Robinson, et al. Mycobacterial Cells Have Dual Nickel-Cobalt Sensors: SEQUENCE RELATIONSHIPS AND METAL SITES OF METAL-RESPONSIVE REPRESSORS ARE NOT CONGRUENT J. Biol. Chem., November 2, 2007; 282(44): 32298 - 32310. [Abstract] [Full Text] [PDF]

				D. S. Touw, C. E. Nordman, J. A. Stuckey, and V. L. Pecoraro Identifying important structural characteristics of arsenic resistance proteins by using designed three-stranded coiled coils PNAS, July 17, 2007; 104(29): 11969 - 11974. [Abstract] [Full Text] [PDF]

				Y. Di and M. J. Tamas Regulation of the arsenic-responsive transcription factor Yap8p involves the ubiquitin-proteasome pathway J. Cell Sci., January 15, 2007; 120(2): 256 - 264. [Abstract] [Full Text] [PDF]

				J. Schelert, M. Drozda, V. Dixit, A. Dillman, and P. Blum Regulation of Mercury Resistance in the Crenarchaeote Sulfolobus solfataricus. J. Bacteriol., October 1, 2006; 188(20): 7141 - 7150. [Abstract] [Full Text] [PDF]

				X. He, M. G. Chen, G. X. Lin, and Q. Ma Arsenic Induces NAD(P)H-quinone Oxidoreductase I by Disrupting the Nrf2{middle dot}Keap1{middle dot}Cul3 Complex and Recruiting Nrf2{middle dot}Maf to the Antioxidant Response Element Enhancer J. Biol. Chem., August 18, 2006; 281(33): 23620 - 23631. [Abstract] [Full Text] [PDF]

				J. Qin, B. P. Rosen, Y. Zhang, G. Wang, S. Franke, and C. Rensing Arsenic detoxification and evolution of trimethylarsine gas by a microbial arsenite S-adenosylmethionine methyltransferase PNAS, February 14, 2006; 103(7): 2075 - 2080. [Abstract] [Full Text] [PDF]

				C. W. Saltikov, R. A. Wildman Jr., and D. K. Newman Expression Dynamics of Arsenic Respiration and Detoxification in Shewanella sp. Strain ANA-3 J. Bacteriol., November 1, 2005; 187(21): 7390 - 7396. [Abstract] [Full Text] [PDF]

				J. Ye, A. Kandegedara, P. Martin, and B. P. Rosen Crystal Structure of the Staphylococcus aureus pI258 CadC Cd(II)/Pb(II)/Zn(II)-Responsive Repressor J. Bacteriol., June 15, 2005; 187(12): 4214 - 4221. [Abstract] [Full Text] [PDF]

				Y. Li, O. P. Dhankher, L. Carreira, D. Lee, A. Chen, J. I. Schroeder, R. S. Balish, and R. B. Meagher Overexpression of Phytochelatin Synthase in Arabidopsis Leads to Enhanced Arsenic Tolerance and Cadmium Hypersensitivity Plant Cell Physiol., December 15, 2004; 45(12): 1787 - 1797. [Abstract] [Full Text] [PDF]

				T. Liu, S. Nakashima, K. Hirose, M. Shibasaka, M. Katsuhara, B. Ezaki, D. P. Giedroc, and K. Kasamo A Novel Cyanobacterial SmtB/ArsR Family Repressor Regulates the Expression of a CPx-ATPase and a Metallothionein in Response to Both Cu(I)/Ag(I) and Zn(II)/Cd(II) J. Biol. Chem., April 23, 2004; 279(17): 17810 - 17818. [Abstract] [Full Text] [PDF]

				L. Song, J. Caguiat, Z. Li, J. Shokes, R. A. Scott, L. Olliff, and A. O. Summers Engineered Single-Chain, Antiparallel, Coiled Coil Mimics the MerR Metal Binding Site J. Bacteriol., March 15, 2004; 186(6): 1861 - 1868. [Abstract] [Full Text] [PDF]

				J. S. Cavet, A. I. Graham, W. Meng, and N. J. Robinson A Cadmium-Lead-sensing ArsR-SmtB Repressor with Novel Sensory Sites: COMPLEMENTARY METAL DISCRIMINATION BY NMTR AND CMTR IN A COMMON CYTOSOL J. Biol. Chem., November 7, 2003; 278(45): 44560 - 44566. [Abstract] [Full Text] [PDF]

				L. Lopez-Maury, F. J. Florencio, and J. C. Reyes Arsenic Sensing and Resistance System in the Cyanobacterium Synechocystis sp. Strain PCC 6803 J. Bacteriol., September 15, 2003; 185(18): 5363 - 5371. [Abstract] [Full Text] [PDF]

				N. Zhou, H. Xiao, T.-K. Li, A. Nur-E-Kamal, and L. F. Liu DNA Damage-mediated Apoptosis Induced by Selenium Compounds J. Biol. Chem., August 8, 2003; 278(32): 29532 - 29537. [Abstract] [Full Text] [PDF]

				B. G. Butcher and D. E. Rawlings The divergent chromosomal ars operon of Acidithiobacillus ferrooxidans is regulated by an atypical ArsR protein Microbiology, December 1, 2002; 148(12): 3983 - 3992. [Abstract] [Full Text] [PDF]

				M. D. Wong, Y.-F. Lin, and B. P. Rosen The Soft Metal Ion Binding Sites in the Staphylococcus aureus pI258 CadC Cd(II)/Pb(II)/Zn(II)-responsive Repressor Are Formed between Subunits of the Homodimer J. Biol. Chem., October 18, 2002; 277(43): 40930 - 40936. [Abstract] [Full Text] [PDF]

				J. S. Cavet, W. Meng, M. A. Pennella, R. J. Appelhoff, D. P. Giedroc, and N. J. Robinson A Nickel-Cobalt-sensing ArsR-SmtB Family Repressor. CONTRIBUTIONS OF CYTOSOL AND EFFECTOR BINDING SITES TO METAL SELECTIVITY J. Biol. Chem., October 4, 2002; 277(41): 38441 - 38448. [Abstract] [Full Text] [PDF]

				S. Li, B. P. Rosen, M. I. Borges-Walmsley, and A. R. Walmsley Evidence for Cooperativity between the Four Binding Sites of Dimeric ArsD, an As(III)-responsive Transcriptional Regulator J. Biol. Chem., July 12, 2002; 277(29): 25992 - 26002. [Abstract] [Full Text] [PDF]

				S. Grkovic, M. H. Brown, M. A. Schumacher, R. G. Brennan, and R. A. Skurray The Staphylococcal QacR Multidrug Regulator Binds a Correctly Spaced Operator as a Pair of Dimers J. Bacteriol., December 15, 2001; 183(24): 7102 - 7109. [Abstract] [Full Text] [PDF]

				K. Hossain, A. A. Akhand, M. Kato, J. Du, K. Takeda, J. Wu, K. Takeuchi, W. Liu, H. Suzuki, and I. Nakashima Arsenite Induces Apoptosis of Murine T Lymphocytes Through Membrane Raft-Linked Signaling for Activation of c-Jun Amino-Terminal Kinase J. Immunol., October 15, 2000; 165(8): 4290 - 4297. [Abstract] [Full Text] [PDF]

				S. Muller, W. H. Miller Jr, and A. Dejean Trivalent Antimonials Induce Degradation of the PML-RARalpha Oncoprotein and Reorganization of the Promyelocytic Leukemia Nuclear Bodies in Acute Promyelocytic Leukemia NB4 Cells Blood, December 1, 1998; 92(11): 4308 - 4316. [Abstract] [Full Text] [PDF]

				C. Thelwell, N. J. Robinson, and J. S. Turner-Cavet An SmtB-like repressor from Synechocystis PCC 6803 regulates a zinc exporter PNAS, September 1, 1998; 95(18): 10728 - 10733. [Abstract] [Full Text] [PDF]

				S. Grkovic, M. H. Brown, N. J. Roberts, I. T. Paulsen, and R. A. Skurray QacR Is a Repressor Protein That Regulates Expression of the Staphylococcus aureus Multidrug Efflux Pump QacA J. Biol. Chem., July 17, 1998; 273(29): 18665 - 18673. [Abstract] [Full Text] [PDF]

				K. Suzuki, N. Wakao, T. Kimura, K. Sakka, and K. Ohmiya Expression and Regulation of the Arsenic Resistance Operon of Acidiphilium multivorum AIU 301 Plasmid pKW301 in Escherichia coli Appl. Envir. Microbiol., February 1, 1998; 64(2): 411 - 418. [Abstract] [Full Text]

				C. Xu and B. P. Rosen Dimerization Is Essential for DNA Binding and Repression by the ArsR Metalloregulatory Protein of Escherichia coli J. Biol. Chem., June 20, 1997; 272(25): 15734 - 15738. [Abstract] [Full Text] [PDF]

				H. Bhattacharjee and B. P. Rosen Spatial Proximity of Cys113, Cys172, and Cys422 in the Metalloactivation Domain of the ArsA ATPase J. Biol. Chem., October 4, 1996; 271(40): 24465 - 24470. [Abstract] [Full Text] [PDF]