The transcriptional repressor protein NsrR senses nitric oxide directly via a [2Fe-2S] cluster

Abstract

The regulatory protein NsrR, a member of the Rrf2 family of transcription repressors, is specifically dedicated to sensing nitric oxide (NO) in a variety of pathogenic and non-pathogenic bacteria. It has been proposed that NO directly modulates NsrR activity by interacting with a predicted [Fe-S] cluster in the NsrR protein, but no experimental evidence has been published to support this hypothesis. Here we report the purification of NsrR from the obligate aerobe Streptomyces coelicolor. We demonstrate using UV-visible, near UV CD and EPR spectroscopy that the protein contains an NO-sensitive [2Fe-2S] cluster when purified from E. coli. Upon exposure of NsrR to NO, the cluster is nitrosylated, which results in the loss of DNA binding activity as detected by bandshift assays. Removal of the [2Fe-2S] cluster to generate apo-NsrR also resulted in loss of DNA binding activity. This is the first demonstration that NsrR contains an NO-sensitive [2Fe-2S] cluster that is required for DNA binding activity.

Figure 1. NsrR and the hmpA genes in S. coelicolor.

(A). Alignment of E. coli IscR with NsrR sequences from Bacillus subtilis (Bsu), E. coli (Eco), S. coelicolor (Sco), Salmonella enterica Typhimurium (Sen), Vibrio vulnificus (Vvu) and Yersinia pestis (Ype). The helix-turn-helix DNA binding motif is highlighted. The three conserved cysteine residues predicted to ligate a [2Fe-2S] cluster are boxed. (B). SCO7427 is the only gene encoding an Rrf2 family member in S. coelicolor. The coding sequence of SCO7427 stops 74 base pairs upstream of the hmpA1 start codon and is predicted to encode an NsrR homologue . Both hmpA genes contain matches to the NsrR consensus for Bacillales and Streptomyces upstream of their translational start codons (boxes) .

Figure 2. CD, UV-visible and EPR spectroscopy of purified NsrR.

(A). The recorded CD spectrum, representing an average of nine individual scans, displays three positive features, λ_max 324, 445, 490 nm, together with two negative features, λ_max 375 and 550 nm, similar to other [2Fe-2S] cluster containing proteins , . The buffer was 75 mM Tris, 425 mM NaCl, 2.5 mM DTT, 2.5% Glycerol, pH 7.5. (B). UV-visible spectra of purified [2Fe-2S] NsrR in 50 mM Tris pH 7.0, 100 mM NaCl buffer or 50 mM Tris pH 7.0, 100 mM NaCl buffer saturated with NO (2 mM), as indicated. UV visible spectroscopy of the purified protein revealed a spectrum characteristic of a [2Fe-2S] cluster containing protein, with major bands at 325 and 420 nm and shoulders present at 460 and 550 nm on the UV visible spectrum [22 Cammack 1980]. The inset spectrum at ×5 magnification shows clearly that the shoulder at 420 nm is lost after exposure to NO (dashed line). (C). X-band EPR spectrum of as isolated NsrR (upper spectrum) and NsrR following exposure to NO (lower spectrum). Measurement conditions were: temperature 10 K; microwave power 2 mW; microwave frequency 9.43755 GHz; modulation amplitude 4 G. NsrR (2.25 uM) was in buffer A and decomposed MAHMA NONOate solutions were prepared in 100 mM Tris-HCl pH 8.0. The purified NsrR protein is EPR silent (top), indicative of an oxidised [2Fe-2S] cluster. Exposure to NO results in a strong signal indicating the formation of a mononuclear dinitrosyl iron complex (bottom, g values are indicated).

Figure 3. DNA binding assays with the hmpA1 and hmpA2 promoters and purified NsrR protein.

(A). Bandshift assay using 200 base pair restriction fragments (20 ng per reaction) carrying the hmpA1 and hmpA2 promoters, as indicated, and purified [2Fe-2S] NsrR in 50 mM Tris pH 7.0, 100 mM NaCl buffer or 50 mM Tris pH 7.0, 100 mM NaCl buffer saturated with NO (2 mM), as indicated. Binding is abolished by addition of NO saturated buffer to purified NsrR. (B). Bandshift assay using a 200 base pair restriction fragment (20 ng per reaction) carrying the hmpA1 promoter with either EDTA∶ferricyanide treated apo-NsrR or holo [2Fe-2S] NsrR as indicated in 50 mM Tris pH 7.0, 100 mM NaCl buffer. The apo-form of the protein is unable to bind to the hmpA1 promoter indicating that the cluster is required for DNA binding activity. (C). Sedimentation equilibrium of oligonucleotides in the presence and absence of purified NsrR. The sedimentation of each sample was monitored at 260 nm and fitted to a single component model as described in Materials and Methods. (Left) Lower panel: absorbance profile of 2 µM hmpA2 and 10 µM NsrR in 50 mM Tris-HCl pH 7.0, 100 mM NaCl after centrifugation at 16,000 (□), 18,000 (O) and 20,000 (Δ) rpm. Upper panel: Residual profile of the difference between the data and fitted curves. (Right) Lower panel: absorbance profile of 2 µM hmpA2 in 50 mM Tris-HCl pH 7.0, 100 mM NaCl after centrifugation at 16,000 (□), 18,000 (O) and 20,000 (Δ) rpm. Upper panel: Residual profile of the difference between the data and fitted curves.