Based on crystal structure and mutation analyses, it was determined that the K64 and R65 residues are crucial for the NikR functions on the nikAp1 promoter in vivo [4]. NikR mediates two levels of repression. Saturation of the high-affinity nickel-binding sites at nickel concentrations in the picomolar range leads to substantial binding to the nik operator and a low level of constitutive repression, while nickel binding to the low-affinity sites at higher nickel concentrations (in the nanomolar range) leads to a large increase in operator binding and strong repression []. His48 and His110 are the residues that participate in the low-affinity Ni(II)-binding response []. In addition to the four high-affinity nickel-binding sites, the crystal structure reveals two binding sites for potassium ions, bridging the metal- and DNA-binding domains []. It is still disputed whether these sites actually resemble the low-affinity binding sites for nickel ions or whether these sites function as potassium-binding sites []. Molecular simulations on the EcNikR structure suggest that binding of NikR to DNA is more favorable when NikR contains K⁺ [], and the allosterically activated DNA-binding conformation of NikR is stabilized by K⁺ []. Moreover, DNA binding of NikR is abolished in the absence of K⁺ []. Reviews: [6]