LAUSR

Significance The highly toxic cadmium ion can cause destructive hazards to living systems by nonspecific and tight binding on functional macromolecules. However, most of the developed cadmium detoxification systems are not sufficient to recognize or detoxify cadmium ions, specifically due to the similar coordination behavior of heavy metal ions in thiolate-rich sites. Here we report that the ultraspecific cadmium regulator CadR has evolved 2 distinct types of functional recognition sites rather than a mono-type thiolate-rich site to achieve outstanding selectivity. The thiolate-rich site I and the adjacent histidine-rich recognition site II are highly associated with transcription activation. This cooperative binding mechanism could improve our understanding of the relationship between the structural dynamics and biological function of metalloregulators. Detoxification of the highly toxic cadmium element is essential for the survival of living organisms. Pseudomonas putida CadR, a MerR family transcriptional regulator, has been reported to exhibit an ultraspecific response to the cadmium ion. Our crystallographic and spectroscopic studies reveal that the extra cadmium selectivity of CadR is mediated by the unexpected cooperation of thiolate-rich site I and histidine-rich site II. Cadmium binding in site I mediates the reorientation of protein domains and facilitates the assembly of site II. Subsequently, site II bridge-links 2 DNA binding domains through ligands His140/His145 in the C-terminal histidine-rich tail. With dynamic transit between 2 conformational states, this bridge could stabilize the regulator into an optimal conformation that is critical for enhancing the transcriptional activity of the cadmium detoxification system. Our results provide dynamic insight into how nature utilizes the unique cooperative binding mechanism in multisite proteins to recognize cadmium ions specifically.