The Structure And Property Of Cadmium Sensing Metalloregulatory Protein CadR

Cadmium is a highly toxic metal element ubiquitously existing in environment and can cause serious damages to human health.Pseudomonas putida CadR belongs to the MerR family regulator,a group of transcriptional activators with N-terminal DNA binding domain and C-terminal effector binding domain that regulate metal ion,radical,and small organic molecule.Comparing to other natural cadmium binding proteins,CadR exhibits an ultra-specific cadmium selectivity because the cadR gene only response to Cd(Ⅱ)and not to Zn(Ⅱ)or Pb(Ⅱ)in vivo.However,the basis underlying cadmium recognition by CadR remains elusive.In this work,we present a comprehensive crystallographic and spectroscopic study of CadR to investigate the cadmium selectivity in CadR.The crystal structure of P.putida CadR and its DNA complex in the absence and presence of Cd(Ⅱ)/Pb(Ⅱ)/Zn(Ⅱ)show CadR is a winged helix homodimer.Each protomer consists of five a helixes and random coils connecting them.Unexpectedly,the crystal structures and titration data reveal that CadR applies two distinct type of metal binding sites.In addition to the common thiolate-rich allosteric site(Site I)adopted by most of metalloregulators,it also contains a histidine-rich metal binding site(Site II)which is structural flexible and unique in the MerR family.Site I is formed by three conserved cysteine residues(Cys77,Cys112 and Cys 119)and one asparagine residue(Asn81).The CdS3O coordination shell in Site I is first reported in natural proteins.It is different from the other two cadmium regulatory proteins CadC and CmtR with the oxygen ligand is a water molecular.Titration experiments show that site Ⅰ binds to Cd(Ⅱ)/Pb(Ⅱ)in high affinity and relative low affinity for Zn(Ⅱ).Metal binding in allosteric site Ⅰ triggers significant conformational changes in C-terminal metal binding domain,and subsequently reorient the DNA binding domain and promoter DNA.Due to a smaller ion size,residue Asn81 is too far away to coordinate with zinc ion in the Zn(II)complexes.Site Ⅱ is formed by two histidine residues(His87 and His90),one glutamic acid residue(Glu62)and a variable ligand from protein(His140/His145)or solvate(SCN-).Metal binding in site Ⅱ can alter the conformation of DBD and bridge-link the two DBDs,thus it stabilizes the distorted protein-DNA complex further.Isomerism in site Ⅱ and the C-terminal tail is observed both in crystal and solution.Site Ⅱ binds to Cd(Ⅱ)and Zn(Ⅱ)with high affinity,while it cannot bind Pb(Ⅱ).The luxAB luciferase reporter data show the CadRH87AH90A mutant with site Ⅱ truncated can hardly response to cadmium ion in vivo.These results suggest that the specific cadmium selectivity of CadR is induced by the cooperative binding mechanism of both sites.