Crystallographic studies of the metal-responsive transcription factor NikR

Abstract

Metal ion homeostasis is critical to the survival of all cells, because requirements for these essential nutrients must be balanced with their toxicity when present at elevated concentrations. Regulation of nickel concentrations in Escherichia coli is achieved via nickel-induced transcriptional repression of the nickel permease complex, NikABCDE, mediated by the NikR metalloregulatory transcription factor. To understand how this takes place at a molecular level, X-ray crystallography was used to determine three- dimensional structures of the NikR. Structures of apo-NikR and Ni⁺²-NikR show an interesting quaternary arrangement of the functional NikR tetramer, consisting of two dimeric Ribbon-Helix-Helix (RHH) DNA-binding domains separated by a tetrameric regulatory domain that binds metal ions (MBD). NikR is the only known metal- responsive member of the RHH family of transcription factors, and the structural relationships between NikR and other RHH proteins are detailed in this thesis. The high- affinity nickel-binding site of NikR is located at the tetramer interface of the MBD, with square-planar coordination by three histidines and a cysteine sidechain. A structure of NikR in complex with a 30 base-pair oligonucleotide containing the nik operator sequence illustrates the dramatic conformational changes associated with DNA binding, shows the nature of the specific and non-specific contacts between NikR and its operator, and reveals a second functionally relevant metal-binding site created at the interface of the RHH domain and MBD. Binding of nickel ions to the high-affinity site of NikR is proposed to increase affinity for the nik operator through a combination of mechanisms.

(cont.) Perhaps the most important of these is by ordering one face of the MBD of NikR, allowing productive interaction with the DNA phosphate backbone in the center of the operator. Additional metal ions further increase the affinity for operator by binding to the site situated at the RHH-MBD domain interface, and perhaps by binding to one of several sites on the surface of NikR identified by soaking crystals in excess Ni⁺².