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. 2024 Oct 15;15(45):18920-18932.
doi: 10.1039/d4sc04618h. Online ahead of print.

Binding of a single nitric oxide molecule is sufficient to disrupt DNA binding of the nitrosative stress regulator NsrR

Jason C Crack  1 , Nick E Le Brun  1
Affiliations

Binding of a single nitric oxide molecule is sufficient to disrupt DNA binding of the nitrosative stress regulator NsrR

Jason C Crack et al. Chem Sci. .

Abstract

The regulatory protein NsrR, a member of the Rrf2 protein superfamily, plays a major role in the cellular response to nitrosative stress in many benign and pathogenic bacteria. The homodimeric protein binds a [4Fe-4S] cluster in each subunit (termed holo NsrR), and represses transcription of genes primarily involved in NO detoxification. Holo NsrR reacts rapidly with multiple NO molecules per [4Fe-4S] cluster, via a complex reaction, with loss of DNA binding and formation of NsrR-bound iron-nitrosyl species. However, the point at which DNA binding is lost is unknown. Here, we demonstrate using surface plasmon resonance (SPR) and native mass spectrometry (MS) that holo NsrR binds the promoter regions of NsrR-regulated genes with promoter-dependent nanomolar affinity, while hemi-apo NsrR (i.e. one cluster per dimer) binds >10-fold less tightly, and the cluster-free (apo) form not at all. Strikingly, native MS provided detailed information about the reaction of NO with the physiologically relevant form of NsrR, i.e. DNA-bound dimeric NsrR. Reaction with a single NO molecule per NsrR dimer is sufficient to abolish DNA binding. This exquisite sensitivity of DNA binding to NO is consistent with the importance of de-repressing NO detoxification systems at the earliest opportunity to minimise damage due to nitrosative stress. Furthermore, the data show that previously characterised iron-nitrosyls, which form at higher ratios of NO to [4Fe-4S], are not physiologically relevant for regulating the NsrR on/off switch.

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Conflict of interest statement

The authors have no conflicts to declare.

Figures

Fig. 1
Fig. 1. Close up view of the [4Fe–4S] NsrR-hmpA1 promoter interface. (A) 2D representation of interaction networks near the [4Fe–4S] cluster. Arg12 from helix 1 connects to Asp8 from the same helix and Val36 from helix 2 of the wHTH domain. Glu85 connects helix 5 to Thr4 and Thr7 of helix 1. Gly37 from helix 2 forms an inter-subunit connection with Asn97 in the cluster-binding loop of the other subunit. These interactions, together with the [4Fe–4S] cluster, correctly position the recognition helix of each subunit for optimal DNA binding (PDB: 7B0C). (B) 3D arrangement of cluster binding loop (white subunit) and wHTH domain from the adjacent subunit (grey subunit) when bound to DNA. The white monomer provides three ligands (Cys 93, 99, 105) to the [4Fe–4S] cluster (in space filling representation) resulting in a well-defined loop structure around the cluster. The fourth ligand, Asp 8, is located close by on the ancillary helix (helix 1) of the wHTH domain from opposite monomer (grey subunit), helping to optimally position the recognition helix (helix 3, grey subunit) for DNA binding. Residues of the recognition helix that ‘read’ the nucleotide sequence are also shown.
Fig. 2
Fig. 2. Native MS [4Fe–4S] NsrR and NsrR-hmpA1 complexes. (A) Deconvoluted spectra of as-isolated and reconstituted [4Fe–4S] NsrR samples (8 μM [4e–4S]), as indicated. As-isolated NsrR is heterogeneous, containing apo, hemi-apo and holo NsrR dimers. Reconstituted NsrR is more homogeneous, containing a high proportion of holo NsrR dimers. (B) Deconvoluted spectra of equivalent samples (8 μM [4e–4S]) treated with hmpA1 DNA (8 μM) resulting in the appearance of [4Fe–4S] NsrR-DNA complexes. Samples were ionised from 100 mM ammonium acetate, pH 8.0 in positive mode. See Table S2 and Fig. S3 for further details.
Fig. 3
Fig. 3. Formation of [4Fe–4S] NsrR-hmpA1 complexes probed by SPR. Analyte binding response of reconstituted [4Fe–4S] NsrR to 29bp hmpA1 promoter region in SPR buffer (black circles) and modified native MS buffer (grey triangles). The data were fitted using a simple binding equation, giving a Kd of 1.2 (±0.2) nM (n = 12) for [4Fe–4S] NsrR in SPR buffer (black line). Inset shows the effect of modified native MS buffer on DNA binding, which was weaker, with a Kd of 0.54 (±0.1) μM (grey line). SPR was performed at 25 °C with SPR buffer (10 mM HEPES, 150 mM NaCl, 0.05% polysorbate 20, pH 7.4) and modified native MS buffer (100 mM ammonium acetate, 0.05% polysorbate 20, pH 8.0).
Fig. 4
Fig. 4. Formation of [4Fe–4S] NsrR-hmpA1 complexes probed by native MS. (A) Deconvoluted mass spectra at selected concentrations of hmpA1 DNA showing the formation of NsrR-hmpA1 complexes from NsrR dimers (8 μM [4Fe–4S]), as indicated. At higher concentrations of hmpA1 non-specific NsrR-hmpA1 complexes appear, involving two DNA molecules. See Fig. S3 for annotation of adducts. (B) Plots of fractional abundance for dimeric, complexed, and non-specific NsrR species as a function of the hmpA1 concentration. Solid lines show fits of the data to a simple sequential binding model, giving a Kd of 3 (±0.1) μM for NsrR-hmpA1 complexes and a Kd of 170 (±70) μM for the non-specific complex. Proteins and complexes were ionised from 100 mM ammonium acetate, pH 8, with positive mode ESI.
Fig. 5
Fig. 5. Formation of NsrR-hmpA1 complexes with heterogeneous NsrR. (A) Deconvoluted mass spectra at selected concentrations of hmpA1 DNA showing the formation of NsrR-hmpA1 complexes from heterogenous NsrR samples (black line, 8 μM [4Fe–4S], 11.4 μM protein, ∼70% loaded). At higher concentrations of hmpA1 hemi-apo and holo NsrR-hmpA1 complexes were observed (red line); see Fig. S3B for annotation of adducts. At low concentrations (≤3 μM) there was a clear preference for holo NsrR dimers (grey lines, as annotated). Apo NsrR exhibited a negligible affinity for DNA, consistent with previous observations. (B) Dimeric region of the spectrum during the same titration, showing the decline of holo NsrR as the titration proceeded. Minor changes in the amount of uncomplexed hemi-apo NsrR dimers were also observed. Proteins and complexes were ionised from 100 mM ammonium acetate, pH 8, with positive mode ESI.
Fig. 6
Fig. 6. SPR kinetics of NsrR-DNA complex formation and dissociation. (A) Comparison of association and dissociation phases for 2 nM [4Fe–4S] NsrR binding to hmpA1 (black line) and hmpA2 (red line) promoter sequences using anaerobic SPR buffer. (B) Association phase form hmpA1 promoter at varying concentrations of [4Fe–4S] NsrR, as indicated. (C) Full association and dissociation phases of data shown in B (black line), together with fits to a bivalent analyte model (red line), giving association and dissociation rate constants and binding affinity, see Table 1. For hmpA2 analysis, see Fig. S6. Analysis temperature was 25 °C.
Fig. 7
Fig. 7. Nitrosylation of NsrR-hmpA1 complex probed by native MS. (A) Comparison of NsrR-hmpA1 complexes before (black line) and after (red line) the addition of NO (as DEA NONOAte). Spectra of NsrR-hmpA1 complex (B) and dimeric NsrR regions (C) are shown in more detail as the in situ nitrosylation proceeds. Following the addition of NONOate (black lines) the spectra gradually change as NO is released (grey lines, increments of ∼0.2 [NO] : [4Fe–4S]). After ∼3.5 [NO] : [4Fe–4S] most of the NsrR-hmpA1 complex was dissociated (red lines). The green line in (C) shows a comparable sample in the absence of DEA NONOate. (D) Average (n = 4) fractional abundance for the NsrR-hmpA1 complex (red squares) and holo NsrR dimers (black circles) as a function of the [NO] concentration. Error bars represent standard deviations. Solid lines represent fits using a simple equilibrium model (see Methods). Samples contained 8 μM [4Fe–4S], 8 μM hmpA1 DNA.
Fig. 8
Fig. 8. Proposed model of NO sensing by holo NsrR. (A) NO binds to the one of the clusters present in holo NsrR, displacing Asp8 to giving a mono-nitrosylated holo NsrR. (B) Displacement of Asp8 by NO likely results in the repositioning of the recognition helix (helix 3), disrupting DNA binding (blue, PDB: 5N08 ref. 34). For comparison, the wHTH domain of NsrR-DNA complex is shown in grey (PDB: 7B0C). (C) Scheme summarizing the mechanism of NsrR NO sensing and regulation. Holo NsrR binds to the promoter sequence upstream of hmpA1 (encoding a flavohemoglobin NO dioxygenase), repressing expression. Endogenous NO is produced by NarG when cytoplasmic NO2− concentrations are elevated. Detection of NO by the [4Fe–4S] cluster of NsrR leads to mono-nitrosyl (and di-nitrosyl) holo NsrR dimers and a loss of DNA binding, allowing the expression of hmpA1 in an NO-dependent manner. HmpA1 converts NO to back to NO2−, lowering the cytoplasmic NO concentration, reinstating holo NsrR-mediated repression. During prolonged nitrosative stress (shown in red) the [4Fe–4S] breaks down, becoming DNIC, RRE, RBS-like and apo-NsrR species. These forms of NsrR (shown in grey) may be clients for FeS cluster assembly, repair, or a target for protein degradation. Repaired (or replaced) [4Fe–4S] NsrR may re-bind the hmpA1 promoter if the cytoplasmic NO concentration is low enough.
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