title: Co(ll)-detection does not follow Kco(ll) gradient: channelling in Co(ll)-sensing.
abstract: The MerR-like transcriptional activator CoaR detects surplus Co(ll) to regulate Co(ll) efflux in a cyanobacterium. This organism also has cytosolic metal-sensors from three further families represented by Zn(ll)-sensors ZiaR and Zur plus Ni(ll)-sensor InrS. Here we discover by competition with Fura-2 that CoaR has KCo(ll) weaker than 7 × 10(-8) M, which is weaker than ZiaR, Zur and InrS (KCo(ll) = 6.94 ± 1.3 × 10(-10) M; 4.56 ± 0.16 × 10(-10) M; and 7.69 ± 1.1 × 10(-9) M respectively). KCo(ll) for CoaR is also weak in the CoaR-DNA adduct. Further, Co(ll) promotes DNA-dissociation by ZiaR and DNA-association by Zur in vitro in a manner analogous to Zn(ll), as monitored by fluorescence anisotropy. After 48 h exposure to maximum non-inhibitory [Co(ll)], CoaR responds in vivo yet the two Zn(ll)-sensors do not, despite their tighter KCo(ll) and despite Co(ll) triggering allostery in ZiaR and Zur in vitro. These data imply that the two Zn(ll) sensors fail to respond because they fail to gain access to Co(ll) under these conditions in vivo. Several lines of evidence suggest that CoaR is membrane associated via a domain with sequence similarity to precorrin isomerase, an enzyme of vitamin B12 biosynthesis. Moreover, site directed mutagenesis reveals that transcriptional activation requires CoaR residues that are predicted to form hydrogen bonds to a tetrapyrrole. The Co(ll)-requiring vitamin B12 biosynthetic pathway is also membrane associated suggesting putative mechanisms by which Co(ll)-containing tetrapyrroles and/or Co(ll) ions are channelled to CoaR.
authors: School/Department of Biological and Biomedical Sciences, Biophysical Sciences Institute, Durham University, DH1 3LE, UK.
authors: Carl J
authors: CJ
authors: Patterson
authors: Rafael
authors: R
authors: Pernil
authors: Samantha J
authors: SJ
authors: Dainty
authors: Buddhapriya
authors: B
authors: Chakrabarti
authors: Clare E
authors: CE
authors: Henry
authors: Victoria A
authors: VA
authors: Money
authors: Andrew W
authors: AW
authors: Foster
authors: Nigel J
authors: NJ
authors: Robinson
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000693:Anaerobiosis / Q000187:drug effects; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D003035:Cobalt / Q000378:metabolism* / Q000494:pharmacology; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005960:Glucosides / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D046939:Synechocystis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D045725:Tetrapyrroles / Q000378:metabolism; D017186:Titrimetry; D014578:Uroporphyrins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005960:Glucosides; D012333:RNA, Messenger; D045725:Tetrapyrroles; D014578:Uroporphyrins; C072476:hydrogenobyrinic acid; D003035:Cobalt; C040358:dodecyl maltoside