Summary:
NikR, "Nickel-responsive repressor," is a tetramer and consists of two domains. The crystal structures of the C-terminal domain, apo-NikR, nickel-activated NikR, and NikR in complex with its operator DNA have been solved [2, 5, 6]. The C-terminal 83 amino acids form the tetrameric core, which carries the high-affinity nickel-binding site and resembles the ACT fold of the ACT (aspartokinase, chorismate mutase, TyrA) family. Two N-terminal domains intertwine to form a dimeric DNA-binding ribbon-helix-helix (RHH) domain [3]. The two RHH DNA-binding domains flank the tetrameric core of the protein [5].
The NikR tetramer contains four high-affinity nickel-binding sites located at the subunit interfaces of the C-terminal domain and additional low-affinity metal-binding sites [5, 7, 8, 9, 10, 11]. The coordination of the nickel ion binding to the high-affinity site depends on binding to DNA. The nickel ion is bound in a four-coordinate planar conformation to NikR in solution and in a six-coordinate conformation to NikR bound to DNA [12].
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
[6, 7, 13]. His48 and His110 are the residues that participate in the low-affinity Ni(II)-binding response
[14].
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
[5]. 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
[5, 15]. Molecular simulations on the EcNikR structure suggest that binding of NikR to DNA is more favorable when NikR contains K
+ [16], and the allosterically activated DNA-binding conformation of NikR is stabilized by K
+ [5, 16]. Moreover, DNA binding of NikR is abolished in the absence of K
+ [17].
Reviews:
[18, 19, 20]
[APPH] Assay of protein purified to homogeneity