A Multiplex Enzymatic Machinery for Cellular Protein S-nitrosylation

Abstract

S-nitrosylation, the oxidative modification of Cys residues by nitric oxide (NO) to form S-nitrosothiols (SNOs), modifies all main classes of proteins and provides a fundamental redox-based cellular signaling mechanism. However, in contrast to other post-translational protein modifications, S-nitrosylation is generally considered to be non-enzymatic, involving multiple chemical routes. We report here that endogenous protein S-nitrosylation in the model organism E. coli depends principally upon the enzymatic activity of the hybrid cluster protein Hcp, employing NO produced by nitrate reductase. Anaerobiosis on nitrate induces both Hcp and nitrate reductase, thereby resulting in the S-nitrosylation-dependent assembly of a large interactome including enzymes that generate NO (NO synthase), synthesize SNO-proteins (SNO synthase), and propagate SNO-based signaling (trans-nitrosylases) to regulate cell motility and metabolism. Thus, protein S-nitrosylation by NO in E. coli is essentially enzymatic, and the potential generality of the multiplex enzymatic mechanism that we describe may support a re-conceptualization of NO-based cellular signaling.

Keywords: OxyR; S-nitrosylase; S-nitrosylation; iron-sulfur cluster; nitrate reductase; nitric oxide; trans-nitrosylase.

Figure 1. Hcp is a Protein S-nitrosylase

(A) During ARN, individual genes in the OxyR regulon differentially regulate intracellular protein nitrosylation levels. The greatest decrease in protein nitrosylation resulted from deletion of hcp as measured by photolysis/chemiluminescence (n=3–21, ±SEM). Dashed line=nitrosylation level in WT. (B) Hcp mediates S-nitrosylation of the majority of SNO-proteins during ARN (biotin switch). HcpF=overexpressed FLAG-tagged Hcp. WT/HcpF=HcpF in WT background. Values are normalized with respect to WT during ARN; N=nitrate, F=fumarate (n=3, ±SEM). Dashed lines indicate lanes removed for clarity. (C) Increased intracellular NO levels (resulting from knockout of NO metabolizing enzymes) do not restore protein S-nitrosylation during ARN in the absence of Hcp. SNO content of lysates was determined by Hg²⁺-coupled photolysis-chemiluminescence (n=6, ±SEM). * p < 0.05 versus WT by Student’s t-test. (D) LpdA is S-nitrosylated in vitro by Hcp. LpdA (1.6 µM) was treated anaerobically with the NO donor DEANO (50 µM; 15 min) and Hcp (1.2 µM) where indicated, and S-nitrosylation was assessed by SNO-RAC. Values are normalized with respect to NO+Hcp, n=2–4±SEM. (E) GAPDH is S-nitrosylated in vitro by Hcp. GAPDH (12.8 µM) was treated as in (D), n=2–4±SEM. (F) S-nitrosylation of OxyR in vivo during ARN (6 hr) is largely dependent upon Hcp (SNO-RAC). n=4, ± SEM, *, differs from WT by ANOVA (p < 0.05). (G) OxyR is S-nitrosylated by Hcp in vitro. OxyR (1.6 µM) was treated as in (D) and (E) above, and S-nitrosylation was detected by SNO-RAC (n=3, ±SEM). (H) Hcp interacts with OxyR in an NO-dependent manner. His-tagged Hcp (HcpHis6) (20 µg) was added to lysates from Δhcp/OxyRF (cells grown anaerobically on nitrate or fumarate). Samples were treated with DEANO (100 µM) where indicated. Following chemical crosslinking, OxyRF was immunoprecipitated and Hcp was detected by western blotting (OxyRF=overexpressed FLAG-tagged OxyR) (n=3, ±SEM).

Figure 2. Metal Clusters and Cysteines Play an Essential Role in Hcp-mediated S-nitrosylation

(A) Following anaerobic treatment of purified recombinant Hcp (1 µM) with the NO donor PROLINONOate (PROLINO) (20 µM), Hcp-bound NO is present as both FeNO and SNO in approximately equal proportions. Protein-bound NO was measured by Hg²⁺-coupled photolysis-chemiluminescence (n=5, ±SEM). (B) Treatment of Hcp with the iron chelator 2’, 2’ bipyridyl (30 min) diminished in vitro S-nitrosylation of OxyR by DEANO, assessed as in Figure 1G; n=3, ±SEM. *p < 0.05 with respect to Hcp + NO by ANOVA. (C) Iron-sulfur clusters mediate Hcp auto-S-nitrosylation. Hcp was treated with 2’, 2’ bipyridyl chelator as in (B) followed by PROLINO (20 µM) and protein-bound NO was measured by Hg²⁺-coupled photolysis-chemiluminescence (UT=untreated; Chel=chelator, n=3, ±SEM). (D) Non-cluster coordinating cysteines in Hcp are not stably S-nitrosylated. Following anaerobic treatment of purified recombinant WT and mutant Hcp (1 µM) with PROLINO (20 µM), protein-bound SNO was measured by Hg²⁺-coupled photolysis-chemiluminescence (n=3, ±SEM). (E) Maintained S-nitrosylation by Hcp requires a redox cycle. OxyR (1.2 µM) was treated anaerobically with DEANO (100 µM) in the presence or absence of Hcp (100 nM), the Hcp reductase Hcr (100 nM) and the electron acceptor NAD⁺ (50 µM). S-nitrosylation of OxyR was assessed by SNO-RAC. Data are presented as SNO-OxyR normalized with respect to total OxyR (n=3, ±SEM).

Figure 3. NO-dependent Formation Within Hcp of an Intramolecular Disulfide, Dimerization and Assembly of a Multi-protein Complex Competent for S-nitrosylation

(A) Hcp-dependent S-nitrosylation during ARN generates a subset of SNO-proteins that are constituents of the Hcp interactome (representing substrates likely S-nitrosylated directly by Hcp), and a larger set of SNO-proteins that are S-nitrosylated through a trans-nitrosylation cascade. The overlap between the Hcp-dependent SNOome and the Hcp interactome is illustrated by a Venn diagram, in which numbers represent individual substrates as identified by mass spectrometry. (B) NO-dependent intramolecular disulfide formation in Hcp. Hcp (1.8 µM) was treated anaerobically with DEANO (200 µM) followed by SDS-PAGE and Coomassie staining. (C) Dimerization of Hcp leads to interactome formation. WT E. coli overexpressing FLAG-tagged Hcp (WT/HcpF) were grown anaerobically either in nitrate (10 mM, 6 hr), or in fumarate followed by treatment with DEANO (100 µM). Lysates were analyzed by non-reducing and reducing SDS-PAGE, and Hcp was visualized by western blotting with anti-FLAG anti-body. Representative gel, n=3. At right, longer exposure of nitrate lane. (D) NO-dependent formation of an intramolecular disulfide in Hcp between Cys102 and Cys155. WT/HcpF were grown as in (C). HcpF was immunoprecipitated followed by non-reducing SDS-PAGE. A single disulfide bond, between Cys102–155, was identified in dimerized but not monomeric Hcp by mass spectrometry (see also Figure S2E). Representative gel, n=3 (UT=untreated; conditions as in 3C). (E) Cys102 and Cys155 are essential for Hcp dimerization and complex formation. WT/HcpF or WT/HcpF C102/155S were grown anaerobically in the presence of either nitrate (N) or fumarate (F) (10 mM). Lysates were analyzed by non-reducing and reducing SDS-PAGE. Hcp was visualized by western blotting with anti-FLAG antibody (n=2). (F) Alkylation of free thiols in Hcp decreases S-nitrosylation of OxyR. Hcp was treated anaerobically with iodoacetamide (IAA) (30 min, 37°C), (followed by removal of IAA=Hcp-IAA) and in vitro S-nitrosylation of OxyR was assessed as in Figure 1G (n=3, ±SEM); * differs from Hcp + DEANO treatment by ANOVA, p < 0.05. (G) Cys102/Cys155 are essential for S-nitrosylation by Hcp. In vitro S-nitrosylation of OxyR was carried out as in (1G). n=3, ±SEM; * differs from Hcp + DEANO treatment by ANOVA, p < 0.05. A dashed line indicates an in-gel cut for clarity. (H) Cys102/Cys155 are essential for S-nitrosylation by Hcp. WT, Δhcp, Δhcp/HcpF, Δhcp/HcpF C102/155S were grown anaerobically in LB medium in the presence of nitrate (10 mM, 2.5 hr). Lysates were subjected to SNO-RAC and SNO proteins were visualized by SDS-PAGE followed by Coomassie staining. (I) Hcp undergoes an NO-dependent conformational change. CD spectra of recombinant Hcp with and without DEANO treatment (Representative spectra, n=3). (J) The NO-induced conformational change is not altered by C102/155S mutation. CD spectra of WT and C102/155S Hcp with and without DEANO treatment. (Representative spectra, n=3). (K) NO-dependent conformational change in Hcp is abrogated by ascorbate. CD spectra of recombinant Hcp with and without DEANO treatment in the presence or absence of ascorbate (Representative spectra, n=3).

Figure 4. Trans-nitrosylases Propagate Hcp-dependent S-nitrosylation

(A) GAPDH-dependent endogenous S-nitrosylation. WT and ΔgapA E. coli were grown anaerobically in either fumarate or nitrate (10 mM, 6 hr) and lysates were subjected to SNO-RAC. SNO-proteins were visualized by Coomassie staining following SDS-PAGE. Representative gel, n=3. Arrows indicate multiple GAPDH-dependent SNO-proteins. (B) GAPDH trans-nitrosylates CodA in vitro. His-tagged SNO-GAPDH (10 µM) was incubated with His-tagged CodA (2.4 µM) (30 min, 37°C). Reaction mixtures were subjected to SNO-RAC and SNO-proteins were visualized by western blotting with an anti-His antibody, n=3, ±SEM. (C) GAPDH trans-nitrosylates NapA in vitro. His-tagged SNO-GAPDH was used to transnitrosylate His-tagged NapA as in (B), n=3, ±SEM. (D) GAPDH interacts directly with CodA in an NO-dependent manner. His-tagged GAPDH (10 µM) was incubated with His-tagged CodA (2.4 µM) in the presence or absence of CysNO (1 mM, 30 min). GAPDH was immunoprecipitated with anti-GAPDH antibody and co-immunoprecipitated CodA was visualized with an anti-His antibody, n=3, ±SEM. (E) GAPDH interacts directly with NapA in an NO-dependent manner. Co-immunoprecipitation of GAPDH with NapA was done as in (D), n=3, ±SEM.

Figure 5. Hcp-Mediated S-Nitrosylation Regulates Cellular Metabolism and is Essential for Protection Against Nitrosative Stress and for Motility During Anaerobic Respiration on Nitrate

(A) LpdA enzymatic activity is inhibited in vitro by Hcp-dependent S-nitrosylation. Following Hcp-dependent S-nitrosylation in vitro (as in Figure 1D), LpdA activity was determined using dihydrolipoamide to lipoamide conversion by monitoring the conversion of NAD⁺ to NADH, at 340 nm (n=3, ±SEM), * p < 0.05 Hcp + NO versus Hcp; Hcp + NO versus NO; Hcp + NO versus UT (untreated). (B) Effect of Hcp knockout on acetyl-CoA levels. Acetyl-CoA was measured in WT and Δhcp cells grown anaerobically on either fumarate or nitrate (10 mM, 6 hr) (n=3 ±SEM). (C) Hcp inhibits nitrate reductase activity. WT and Δhcp E. coli were grown for 4 hr on nitrate anaerobically to induce nitrate reductase activity, washed and re-exposed to nitrate (5 mM). Nitrite formed was measured with the Greiss assay (n=4, ±SEM), * p < 0.05 WT versus Δhcp. (D) ARN confers Hcp-dependent protection from exogenous nitrosative stress. E. coli cultures were started in minimal medium including 10 mM fumarate or 10 mM nitrate at A600 of 0.1. Where indicated sodium nitroprusside (NP; 2 mM) was added. Growth was followed spectrophotometrically at A600 (n=3, ±SEM). * p < 0.05 WT vs Δhcp. (E) Hcp is essential for E. coli motility during ARN. WT and Δhcp were introduced onto soft agar plates that were supplemented with either fumarate or nitrate (20 mM). Following growth at 37°C for 18 hrs under anaerobic conditions the plates were photographed under white light. Representative results, n=5. (F) Hcp is essential for E. coli motility during ARN. Quantification of E. coli motility following assay described in Figure 5E (n=5, ±SEM). * p < 0.05 WT (nitrate) vs Δhcp (nitrate). (G) A schematic summary of the Hcp-based enzymatic machinery mediating de novo protein S-nitrosylation and S-nitrosylation signaling cascades in E. coli. NarGHI generates NO (NO synthase activity); Hcp converts NO to SNO (SNO-synthase) and trans-nitrosylates binding partners within the Hcp interactome (S-nitrosylase activity), including additional trans-nitrosylases (e.g. SNO-GAPDH) that further propagate SNO-based signals. The Cys-coordinated iron cluster that mediates auto-S-nitrosylation is depicted schematically in the second section. The Hcp dependent interactome comprises over 100 proteins including enzymes (e.g. NarGHI, LpdA) and transcription factors (OxyR) regulating cell growth and metabolism, and conferring protection against nitrosative stress.