Ni(II) and Co(II) Sensing by Escherichia coli RcnR

Abstract

Escherichia coli RcnR and Mycobacterium tuberculosis CsoR are the founding members of a recently identified, large family of bacterial metal-responsive DNA-binding proteins. RcnR controls the expression of the metal efflux protein RcnA only in response to Ni(II) and Co(II) ions. Here, the interaction of Ni(II) and Co(II) with wild-type and mutant RcnR proteins is examined to understand how these metals function as allosteric effectors. Both metals bind to RcnR with nanomolar affinity and stabilize the protein to denaturation. X-ray absorption and electron paramagnetic resonance spectroscopies reveal six-coordinate high-spin sites for each metal that contains a thiolate ligand. Experimental data support a tripartite N-terminal coordination motif (NH2-Xaa-NH-His) that is common for both metals. However, the Ni(II)- and Co(II)-RcnR complexes are shown to differ in the remaining coordination environment. Each metal coordinates a conserved Cys ligand but with distinct M-S distances. Co(II)-thiolate coordination has not been observed previously in Ni(II)-/Co(II)-responsive metalloregulators. The ability of RcnR to recruit ligands from the N-terminal region of the protein distinguishes it from CsoR, which uses a lower coordination geometry to bind Cu(I). These studies facilitate comparisons between Ni(II)-RcnR and NikR, the other Ni(II)-responsive transcriptional regulator in E. coli, to provide a better understanding how different nickel levels are sensed in E. coli. The characterization of the Ni(II)- and Co(II)-binding sites in RcnR, in combination with bioinformatics analysis of all RcnR/CsoR family members, identified a four amino acid fingerprint that likely defines ligand-binding specificity, leading to an emerging picture of the similarities and differences between different classes of RcnR/CsoR proteins.

Biochemical studies on Helicobacter pylori arginase: Insight into the difference in activity compared to other arginases

“…Analysis of our data showed several unique features of this enzyme compared to other arginases; (i) cooperativity in the pH range 6.2-9.8, (ii) self-association and activation with increasing concentrations of the protein, (iii) lower proportion of dimer, and (iv) dimeric protein is associated with higher catalytic activity. Furthermore, we show that Co 21 ions play an important role in the local tertiary structure of the protein than Mn 21 . Overall, we describe several factors that are associated with the biochemical differences between H. pylori and other arginases.…”

“…Arginase (EC 3.5.3.1) is a binuclear Mn 21 -metalloenzyme that catalyzes the conversion of L-arginine to L-ornithine and urea (1). The enzyme is widely present throughout the evolutionary spectrum including bacterium, plant, yeast, and vertebrates, and is important for L-arginine homeostasis (2-4).…”

“…The enzyme is widely present throughout the evolutionary spectrum including bacterium, plant, yeast, and vertebrates, and is important for L-arginine homeostasis (2-4). All arginases exhibit higher catalytic activity with Mn 21 as a metal cofactor except the Helicobacter pylori (H. pylori), which shows higher activity with Co 21 . These proteins usually display optimum activity at an alkaline pH 9-11 (5,6).…”

Metal Selectivity of the Escherichia coli Nickel Metallochaperone, SlyD

“…In the absence of Ni(II) supplementation, the distinct effects of ΔslyD on the activities of NikR and RcnR could be explained by the higher affinity of NikR for Ni(II) (33,44), such that it is more responsive than RcnR to changes in low levels of available metal. However, upon Ni(II) supplementation a marked change in rcnA expression is observed, indicating that sufficient Ni(II) enters the cell to cause de-repression by RcnR, but the slyD deletion still has no impact.…”

“…In addition, Co(II) was of particular interest to us because two other proteins involved in nickel homeostasis, RcnR and RcnA, also contribute to cobalt homeostasis in E. coli (33)(34)(35). To determine whether SlyD could coordinate Co(II), a direct cobalt titration of apo SlyD was analyzed via electronic absorption spectroscopy ( Figure 5).…”