HK Binding Interface in Vibrio cholerae by

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Mapping the H-NOX/HK binding interface in Vibrio cholerae by hydrogen/deuterium exchange mass spectrometry Yirui Guo, Anthony T. Iavarone, Matthew M. Cooper, and Michael A Marletta Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00027 • Publication Date (Web): 19 Feb 2018 Downloaded from http://pubs.acs.org on February 25, 2018

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Biochemistry

Title: Mapping the H-NOX/HK binding interface in Vibrio cholerae by hydrogen/deuterium exchange mass spectrometry Authors: Yirui Guo1, Anthony T. Iavarone1,2, Matthew M. Cooper3 and Michael A. Marletta1,2,3,* 1 California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720 2 Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720 3 Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720 * Corresponding author, email: [email protected] Abstract Heme-nitric oxide/oxygen binding (H-NOX) proteins are a group of hemoproteins that bind diatomic gas ligands such as nitric oxide (NO) and oxygen (O2). H-NOX proteins typically regulate histidine kinases (HK) located within the same operon. It has been reported that NO-bound H-NOXs inhibit cognate histidine kinase autophosphorylation in bacterial H-NOX/HK complexes, however, a detailed mechanism of NO-mediated regulation of the H-NOX/HK activity remains unknown. In this study, the binding interface of Vibrio cholerae (Vc) H-NOX/HK complex was characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS) and further validated by mutagenesis, leading to a new model for NO-dependent kinase inhibition. A conformational change in Vc H-NOX introduced by NO generates a new kinase-binding interface, thus locking the kinase in an inhibitory conformation.

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Introduction Nitric oxide (NO) is an important signaling molecule that is involved in several physiological functions. The primary NO receptor in eukaryotes is soluble guanylate cyclase (sGC)1. NO binding to the N-terminal heme domain of sGC activates guanylate cyclase activity, which converts guanosine 5¢-triphosphate (GTP) to guanosine 3¢,5¢-cyclic monophosphate (cGMP). cGMP can then act as a second messenger inside the cell that initiates a signaling cascade regulating downstream proteins including cGMP-dependent kinases and cGMP-gated ion channels1, 2. The molecular mechanism of NO-induced sGC activation is not fully understood, but it has been demonstrated that an important step in activation involves a conformational change caused by NO binding to the N-terminal heme domain2-4. Homologs of the sGC N-terminal heme domain are found in prokaryotes with the ability to bind NO and/or O2 and are termed H-NOX (Heme-Nitric oxide/OXygen) proteins5-7. They are often found as standalone proteins within the same operon with signaling partners such as histidine kinases (HK) and diguanylate cyclases. Alternatively, they serve as sensor domains in methyl-accepting chemotaxis proteins (MCPs). In both cases, H-NOXs regulate the activity of their associated signaling partner through different ligation states of the H-NOX heme8, 9.

Figure 1

N

H-NOX

Histidine kinase

O

H-NOX

ATP ADP

HK

P

HnoB

P

pGpG

Response regulator

c-di-GMP

biofilm

Figure 1. Overview of NO signaling in H-NOX/HK network. In the absence of NO, the H-NOX-associated HK autophosphorylates a histidine residue and then transfers the phosphoryl group to downstream response regulator (RR) HnoB, which facilitates the conversion of cyclic-di-GMP into diguanylic acid (pGpG). Upon NO binding to the H-NOX, HK autophosphorylation and the subsequent phosphotransfer is inhibited, leading to the accumulation of cyclic-di-GMP and biofilm formation.


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Biochemistry

The H-NOXs of obligate anaerobes contain a hydrogen-bonding network in the distal ligand pocket, which allows the protein to form stable complexes with both NO and O26, 10. This type of H-NOX domain is fused to MCPs and is likely involved in the regulation of chemotactic responses to O211. However, H-NOXs found in aerobes and facultative anaerobes often selectively bind NO and the signaling partner is usually a histidine kinase (HK) or diguanylate cyclase 12-14. In the case of Shewanella oneidensis (So), a model system used to understand H-NOX/HK function, the H-NOXassociated HK autophosphorylates a conserved histidine residue in the absence of NO. The phosphoryl group is then transferred to cognate response regulators (RR) including HnoB, a phosphodiesterase that hydrolyzes cyclic-di-GMP into diguanylic acid (pGpG)15 (Figure. 1). Upon NO binding to the H-NOX, HK autophosphorylation and the subsequent phosphotransfer is inhibited, leading to the accumulation of cytoplasmic cyclic-di-GMP levels and subsequent biofilm formation, perhaps as a defense mechanism against NO. Biophysical and biochemical characterization of the So H-NOX/So HK suggest that the protein complex can be formed regardless of the So H-NOX ligation states, but the NO-bound H-NOX increases the binding affinity for HK13, 16. There is therapeutic interest in H-NOX/HK signaling in pathogens such as Vibrio cholerae (Vc), as this pathway may be involved in resistance to high NO concentrations derived from the host innate immune response17, 18. Previous studies have shown that Vc HK activity is inhibited by NO-bound Vc H-NOX, similar to the So H-NOX/So HK complex, and may play a role in controlling biofilm formation during colonization in the gut18-20 (Figure. 1). However, the mechanism for NO-induced inhibition has only been partially characterized 16, 19. In this study, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used to directly map the binding interface between Vc H-NOX and its cognate kinase in response to NO, generating a new model for NO-dependent HK inhibition. These results could shed light on the general mechanism of H-NOX/HK regulation and provide potential implications for future anti-biofilm drug design. Materials and Methods Protein expression and purification For bacterial expression, full length Vc H-NOX (1-181) (gene name: VCA0720) was cloned into a pET20b vector with a TEV-cleavable His6 tag at the C-terminus. Protein was expressed from RP523(DE3) cells21, grown in Terrific broth (Research Products International) at 37 °C in the presence of 100 μg/mL ampicillin. Cells were induced with 1 mM IPTG at OD600 of 0.7. After overnight growth at 18 °C, cells were harvested by spinning at 4°C, 4700 ×g for 25 min. The pellet was re-suspended in buffer (50 mM phosphate, pH 8.0, 300 mM NaCl, 5% glycerol) and stored at –80°C. Vc H-NOX variants were generated by quick-change mutagenesis from pET20b Vc HNOX TEV-His wild type and expressed from BL21(DE3) cells in a similar manner with 500 μM FeCl3 added prior to the subculture and 1 mM 5-aminolevulinic acid hydrochloride (Acros Organics) added prior to induction. Protein was purified by affinity chromatography (His60 Ni superflow resin, Clontech) and the C-terminal His6 tag was cleaved by TEV protease. Vc H-NOX used in HDX-MS experiment was


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further purified by size-exclusion chromatography (Superdex 75, GE Healthcare) in 50 mM HEPES, pH 8.0, 150 mM NaCl, 2 mM TCEP and 5% glycerol. Purified Vc HNOX was transferred to a glove bag (Coy Laboratory Products) under an argon/hydrogen (95:5%) atmosphere, at ~25 °C. Vc H-NOX was fully oxidized (λmax 403 nm) using 1-2 equivalents of potassium ferricyanide (K3Fe(CN)6) followed by buffer exchange through a PD10 desalting column (GE healthcare) to remove excess K3Fe(CN)6. The protein was then fully reduced to the Fe(II) state (λmax 430 nm) using 1 mM sodium dithionite (final concentration). Residual dithionite was removed by exchanging the protein into fresh buffer (50 mM HEPES pH 8.0, 50 mM NaCl, 2 mM TCEP and 5% v/v glycerol)22. Full length Vc HK (1-315) (gene name: VCA0719) was cloned into a pHisMBP parallel vector with an N-terminal maltose binding protein (MBP) or a pHisGST parallel vector with an N-terminal glutathione-S-transferase (GST) tag and expressed in BL21(DE3) cells in Terrific broth in the presence of 100 μg/mL ampicillin23. Protein was induced at OD600 ~0.6 with 500 μM IPTG and harvested after overnight growth at 18 °C. Vc HK truncations were cloned into pHisGST parallel vector and expressed in the same manner. Vc HK mutations were generated by quick-change mutagenesis from full length HisGST Vc HK wild type. The pellet was re-suspended in buffer (50 mM Tris, pH 8.0, 50 mM NaCl, 10 mM MgCl2, 5% v/v glycerol) and stored at –80 °C. Purification was conducted using affinity chromatography (His60 Ni superflow resin). Tag-free protein was generated by TEV cleavage of the HisMBP tag overnight at 4 °C and the cleaved tag was removed by Amylose Resin (NEB). Protein was further purified by size-exclusion chromatography (Superdex 200, GE Healthcare) in 50 mM HEPES, pH 8.0, 50 mM NaCl, 10 mM MgCl2, 2 mM TCEP and 5% v/v glycerol. Size-exclusion chromatography multi-angle light scattering (SEC-MALS) Samples were injected onto a Superdex 200 10/300 SEC column (GE Healthcare) equilibrated with 50 mM HEPES pH 8.0, 50 mM NaCl, 10 mM MgCl2, 2 mM TCEP and 5% v/v glycerol. Protein was eluted with 0.5 mL/min and detected with in-line DAWN HELEOS II light scattering and Optilab rEX refractive index detectors (Wyatt Technology). All steps were performed at room temperature (22 °C). Kinase assays Vc HK (4 µM) was incubated with Vc H-NOX (4, 8, 20, 40, or 100 μM) treated with DEA NONOate (0 or 1 mM) in a final volume of 20 μL for 30 min inside a glove bag under an argon/hydrogen (95:5%) atmosphere at ~25 °C. Assays were performed outside the glove bag by adding a mixture of 2.5 μCi [γ-32P] ATP (PerkinElmer) and 1 mM ATP (final concentration), and quenched after 30 min by SDS loading dye with 20 mM EDTA (final concentration). Reactions were analyzed by SDS-PAGE as described previously10. Dried gels were exposed for 2 hours on a phosphor imager plate (GE Healthcare) and imaged using a Typhoon Trio (GE Healthcare) set to storage phosphor mode at 200 μm resolution. Phosphorylated Vc HK bands were quantified using ImageQuant software. Experiments were performed in two independent replicates and averaged.


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Biochemistry

Homology models All homology models were generated using the SWISS-MODEL server24-26. The Vc HNOX model was generated using the crystal structure of NO-bound H-NOX from Shewanella oneidensis (PDB 4U9B)27 as a template. The Vc HK model was generated using the crystal structure of human bZIP transcription factor c-Fos-c-Jun (PDB 1FOS)28 as a template for the N-terminal domain (residue 7-53) and the crystal structure of a sensor histidine kinase YF1 (PDB 4GCZ)29 as a template for part of the N-terminal domain, the DHp and CA domains (residues 32-311). Hydrogen/deuterium exchange mass spectrometry (HDX-MS) Samples were prepared in triplicate with exchange time points of 0, 30 and 7200 seconds. Vc H-NOX Fe(II) or Vc H-NOX Fe(II)–NO (10 μL, 200 μM ) was incubated with 10 μL, 200 μM or 600 μM Vc HK at room temperature for 1 hour in a glove bag prior to H/D exchange. To initiate exchange, the Vc H-NOX/Vc HK mixture was diluted with 180 μL of D2O-based HDX buffer (50 mM HEPES, pD 8.0, 50 mM NaCl, 10 mM MgCl2, 2 mM TCEP and 5% v/v glycerol). At each time point, a 50 μL aliquot of the reaction mixture was quenched with 7 μL of 2.5% v/v trifluoroacetic acid (TFA) to reach final pH of 2.5 and flash-frozen in liquid nitrogen. Samples were then thawed and digested with 50 μL immobilized pepsin (Pierce, pre-washed in acidified H2O-based HDX buffer) and 6 μL of 8 M guanidine hydrochloride on ice for 5 min. Pepsin was removed using a 0.2 μm spin filter. Digested samples were flashfrozen in liquid nitrogen and stored on dry ice until analysis by mass spectrometry. To control for back-exchange, 10 μM Vc H-NOX and Vc HK were pepsin-digested in H2O-based HDX buffer as described above and lyophilized. The peptide mixture was then dissolved in D2O-based HDX buffer with pD adjusted to 9.0 by NaOD (Cambridge Isotopes). The solution was subsequently incubated at 90 °C for 2 hours followed by TFA quenching. HDX data was analyzed using HDX WorkBench30. Values were normalized for 100% D2O. Back-exchange of each peptide ranged between 0– 20% for Vc H-NOX and 0–30% for Vc HK. Liquid chromatography-tandem mass spectrometry for peptide identification Acetonitrile, formic acid (Optima grade, 99.9%, Thermo Fisher Scientific), and water purified to a resistivity of 18.2 MΩ·cm (at 25 °C) using a Milli-Q Gradient ultrapure water purification system (Millipore, Billerica, MA) were used to prepare mobile phase solvents for liquid chromatography-mass spectrometry (LC-MS). To identify peptides resulting from proteolytic digestion, samples of pepsin-digested proteins were analyzed using a Thermo Dionex UltiMate3000 RSLCnano liquid chromatograph that was connected in-line with an LTQ-Orbitrap-XL mass spectrometer equipped with an electrospray ionization (ESI) source (Thermo Fisher Scientific). The LC was equipped with a C18 analytical column (Acclaim® PepMap 100, length: 150 mm, inner diameter: 0.075 mm, particle size: 3 µm, Thermo). Solvent A was 99.9% water/0.1% formic acid and solvent B was 99.9% acetonitrile/0.1% formic acid (v/v). The elution program consisted of isocratic flow at 1% B for 4 min, a linear gradient to 30% B over 38 min, isocratic flow at 95% B for 6 min, and isocratic flow at 1% B for 12 min, at a flow rate of 300 nL/min. The column exit was connected to the ESI source of the mass spectrometer using


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polyimide-coated, fused-silica tubing (inner diameter: 20 µm, outer diameter: 280 µm, Thermo Fisher Scientific). Full-scan mass spectra were acquired in the positive ion mode over the range m/z = 350 to 1800 range using the Orbitrap mass analyzer, in profile format, with a mass resolution setting of 60,000 (at m/z = 400, measured at full width at half-maximum peak height). In the data-dependent mode, the eight most intense ions exceeding an intensity threshold of 50,000 counts were selected from each full-scan mass spectrum for tandem mass spectrometry (MS/MS) analysis using collision-induced dissociation (CID). Real-time dynamic exclusion was enabled to preclude re-selection of previously analyzed precursor ions. Data acquisition was controlled using Xcalibur software (version 2.0.7, Thermo). Raw data were searched against the amino acid sequences of the full-length proteins using Proteome Discoverer software (version 1.3, SEQUEST, Thermo) to identify peptides from the MS/MS spectra. Liquid chromatography-mass spectrometry for hydrogen/deuterium exchange measurements. Deuterated, pepsin-digested protein samples from HDX experiments were analyzed using an Agilent 1200 series LC (Santa Clara, CA) that was connected in-line with the LTQ-Orbitrap-XL mass spectrometer (Thermo) described above. The LC was equipped with a reversed-phase analytical column (Viva C8, length: 30 mm, inner diameter: 1.0 mm, particle size: 5 µm, Restek, Bellefonte, PA) and guard pre-column (C8, Restek). Solvent A was 99.9% water/0.1% formic acid and solvent B was 99.9% acetonitrile/0.1% formic acid (v/v). Each sample was thawed immediately prior to injection. The elution program consisted of a linear gradient from 5% to 10% B over 1 min, a linear gradient to 40% B over 3.5 min, a linear gradient to 95% B over 4.5 min, isocratic conditions at 95% B for 3 min, a linear gradient to 5% B over 0.5 min, and isocratic conditions at 5% B for 5.5 min, at a flow rate of 300 µL/min. The column compartment was maintained at 4 °C and lined with towels to absorb atmospheric moisture condensation. The column exit was connected to the ESI source of the mass spectrometer using PEEK tubing (inner diameter: 0.005”, outer diameter: 1/16”, Agilent). Mass spectra were acquired in the positive ion mode over the range m/z = 350 to 1800 using the Orbitrap mass analyzer, in profile format, with a mass resolution setting of 100,000 (at m/z = 400). Data acquisition was controlled using Xcalibur software (version 2.0.7, Thermo). Pulldown assays For pulldown experiments, purified HisGST Vc HK (4 μM) and Vc H-NOX (18 or 36 μM) were incubated in sealed tubes with 15 μL of Pierce glutathione agarose (Thermo Fisher Scientific) in pulldown buffer (50 mM HEPES, pH 8.0, 50 mM NaCl, 10 mM MgCl2, 2 mM TCEP and 5% v/v glycerol) overnight at 4 °C. Beads were washed twice with buffer before elution. Protein was eluted using 20 μL of 6x SDS loading dye and was resolved by SDS-PAGE. Band intensity was quantified using ImageJ31. UV-Vis spectra for all H-NOX mutants were obtained to ensure that ligand binding properties were the same as the wild type protein (data not shown).


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Biochemistry

Results and discussion Vc H-NOX/Vc HK complex formation Vc H-NOX consists of a single H-NOX domain with a heme cofactor for gas sensing. The cognate Vc HK is a multi-domain protein consists of an N-terminal domain (NTD, mostly α-helical based on secondary structure prediction), a dimerization domain (DHp) with the phosphoacceptor histidine (H71) and a catalytic domain (CA) at the C-terminus that binds ATP and catalyzes phosphotransfer (Figure. 2a). To test the ability of Vc H-NOX to regulate Vc HK autophosphorylation, kinase activity assays were performed with various concentrations of the H-NOX in both the absence and presence of NO. Consistent with previously published results19, Vc HK is inhibited by Vc H-NOX Fe(II)–NO in a concentration-dependent manner. Similar to the So H-NOX/So HK pair, unliganded Vc H-NOX starts to inhibit the kinase activity at high concentrations10, 16, 19 (Figure. 2b, Figure. S1). It has been shown with other H-NOXs that H-NOX/HK complex formation is not dependent on NO13, 16. To test if this is the case for the Vc H-NOX/Vc HK complex, size-exclusion chromatography multi-angle light scattering (SEC-MALS) experiments were performed to analyze the oligomerization states of each protein, as well as the binding stoichiometry of the complex. As shown in Figure 2c, Vc HNOX Fe(II) elutes as a single peak (Vr = 16 mL) with a calculated molar mass (~20 kDa) very close to the theoretical molar mass (21.6 kDa), demonstrating that Vc HNOX is a monomer in solution. Elution of Vc HK (Vr = 12.5 mL) provides a calculated molar mass of approximately 62 kDa, consistent with a Vc HK dimer (theoretical monomeric molar mass: 35 kDa) (Figure 2c). The Vc H-NOX/Vc HK complex formed with excess H-NOX exhibited two main elution peaks with retention volumes of 12 mL and 16 mL, respectively. The former has a calculated molar mass of approximately 100 kDa, consistent with a HK dimer and two H-NOX monomers; the latter has a calculated molar mass around 20 kDa, consistent with the unbound Vc H-NOX. This result confirms that the Vc H-NOX/Vc HK complex is a heterotetramer with 1:1 stoichiometry of the two proteins and that the formation of this complex does not require NO (Figure 2c). Mapping NO-induced conformational change of Vc H-NOX As a first step towards understanding NO-dependent inhibition of Vc HK autophosphorylation, the NO-induced conformational changes of Vc H-NOX were mapped using hydrogen/deuterium exchange mass spectrometry (HDX-MS). Deuterium incorporation (%D) was measured for Vc H-NOX Fe(II) and Vc H-NOX Fe(II)–NO. The H/D exchange perturbation (Δ%D = %DH-NOX Fe(II)–NO – %DH-NOX Fe(II)) was mapped onto a Vc H-NOX homology model (Figure 3a,b; Figure S2). As shown in Figure 3b, NO binding led to an extensive increase in H/D exchange relative to Vc HNOX Fe(II) (Δ%D >0), suggesting that NO binding induces global conformational changes in Vc H-NOX that expose previously buried residues to solvent. The most


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Biochemistry

pronounced H/D exchange increase is observed on residues around the hemebinding pocket, especially the αF helix where the heme-ligating histidine (His 104) Figure 2 a

1

181

Vc H-NOX FL

H-NOX 1

59 60

Vc HK FL

al on e

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H

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1x

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~100 kDa

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0.2 ~62 kDa

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0.1 0.0

Figure 2. NO signaling in Vc H-NOX/Vc HK complex. a) Domain structure of Vc H-NOX and Vc HK. Vc H-NOX is a single domain protein with 181 residues. Vc HK has three domains: an N-terminal domain (NTD, residues 1–59), a dimerization domain (DHp, residues 60–160) and a catalytic domain (CA, residues 161– 315). b) Vc HK autophosphorylation inhibition by Vc H-NOX. Vc HK was incubated with varying concentrations of of Vc H-NOX in the absence or presence of NO. Quantified band intensity in below graph shows kinase activity could be inhibited by Vc H-NOX Fe(II)–NO or high concentration of Vc H-NOX Fe(II). * indicates P 0) around the αF helix and the heme pocket. c) Comparison of Vc H-NOX Fe(II)–NO heat map model to crystal structures of So H-NOX Fe(II) (PDB: 4U99) and So H-NOX Fe(II)–NO (PDB: 4U9B). The highly perturbed Vc H-NOX αF helix region likely undergoes a 90° rotation, similar to that of So H-NOX Fe(II) upon NO binding.

Mapping the Vc H-NOX/Vc HK binding interface in the absence of NO


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Reduced H/D exchange (Δ%D < 0) is expected in areas near the direct binding interface between two proteins due to limited access to solvent32, 33. Here, HDX-MS was used to map the binding interface between Vc H-NOX and Vc HK in the absence of NO. The H/D exchange perturbation of H-NOX Fe(II) in the absence and presence of HK was mapped on the Vc H-NOX homology model (Δ%D = %DH-NOX Fe(II)/HK – %DH-NOX Fe(II)) (Figure 4a; Figure S3). As shown in the model, peptides with reduced H/D exchange (Δ%D < 0) are primarily localized on the N-terminal helices (αA, αB and αC) of Vc H-NOX, suggesting that this is the main subdomain that interacts with Vc HK. This is consistent with a 15N-1H HSQC NMR study of Shewanella woodyi H-NOX and its cognate cyclic-di-GMP-processing enzyme (HaCE), although a CO ligand was used to avoid NO-induced line broadening14. Additionally, the H/D exchange perturbation for Vc HK was investigated. Upon H-NOX binding, there is a large protected area (Δ%D < 0) located at the N-terminal domain of the HK protein (Δ%D = %DH-NOX Fe(II)/HK – %DHK), which is consistent with observations in the So H-NOX/So HK complex16 (Figure 4b; Figure S4). In order to further probe details of the protein/protein interaction, the H/D exchange behavior of each peptide at the binding interface was investigated. For peptides with significant H/D exchange perturbation, two types of exchange behavior were observed over the course of the assay (30 s vs 7200 s). This information was used to identify residues that make direct contact with each other between H-NOX and HK, assuming that amino acids on the same binding interface would have similar H/D exchange behavior (Figure 5). The first type of exchange behavior is typified by Vc H-NOX peptide 27-48, which is located at a loop region between αB and αC (Figure 5a). When HK is absent, this region is largely exposed to solvent, resulting in high deuteration in 30 s (%D = ~70%) and a 5% increase at 7200 s (%D = ~75%). When HK is present, a slower exchange rate is observed that the deuteration is only ~50% at 30 s but increases to ~75% at 7200 s, which is about the same level as in H-NOX alone condition. This interaction must be weak such that the peptide equilibrates with the deuterium content of the solvent over time. A few overlapping peptides located on the HK Nterminus (residues 4-12) were identified with similar H/D exchange behavior and they are likely making direct contacts with this loop region of Vc H-NOX (residues 27-48) in a similar manner (Figure 5a). The second type of exchange behavior is represented by Vc H-NOX peptides 7-12 and 21-26 (helices αA and αB) (Figure 5b). In the case of peptide 7-12, the residues exhibit the same level of deuterium incorporation (%D = ~15%) at 30 s in both absence and presence of HK, but the peptide deuteration is slowed down by HK binding over time (%DH-NOX Fe(II)/HK = ~30%, %DH-NOX = ~70%). This indicates a stronger interaction in that the Vc H-NOX residues within this interaction surface are better protected from the solvent and equilibrate with the deuterium at a much


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Figure 4 a

180°

Less exchange Δ%D -18

More exchange +18

b N

N

N-terminal domain

N

180° CA

DHp/CA

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Biochemistry

His

His71

Figure 4. Mapping the Vc H-NOX/Vc HK binding interface in the absence of NO. Less exchange More exchange Δ%D -18 +18 a) Heat map of Vc HK-induced H/D exchange perturbation on Vc H-NOX homology model (Δ%D = %DH-NOX Fe(II)/HK – %DH-NOX Fe(II)). Vc H-NOX helices αA–C showed decreased exchange (Δ%D < 0), indicating that this area is protected by Vc HK binding. b) Heat map of Vc HNOX-induced H/D exchange perturbation on Vc HK homology model (Δ%D = %DH-NOX Fe(II)/HK – %DHK). The N-terminal domain of Vc HK exhibited decreased exchange (Δ%D < 0), indicating that this area is protected by the H-NOX. A schematic structure of Vc HK is shown on the right with histidine 71 highlighted in red.


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Biochemistry

Figure 5 a Vc H-NOX Fe(II) Vc H-NOX Fe(II)/Vc HK Vc H-NOX peptide 27-48 100

%D

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residues 27-48

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Vc HK Vc H-NOX Fe(II)/Vc HK Vc HK peptide 11-19 (residues 4-12)

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Figure 5. Comparison of peptide H/D exchange behavior. a) Top panel: H/D exchange behavior of H-NOX peptide 27–48 (highlighted on Vc H-NOX homology model); bottom panel: H/D exchange behavior of multiple overlapping peptides on HK residues 10–20 (highlighted on Vc HK homology model). This group of peptides represents the first type of exchange behavior with large Δ%D at 30 s and small Δ%D at 7200 s. b) Top panel: H/D exchange behavior of H-NOX peptides 7–12 and 21–26 (highlighted on Vc H-NOX homology model); bottom panel: hydrogen/deuterium exchange behavior of multiple overlapping peptides on Vc HK residues 25–43 (highlighted on Vc HK homology model). This group of peptides represents the second type of exchange behavior with small Δ%D at 30 s and large Δ%D at 7200 s.


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Biochemistry

slower rate. Overlapping Vc HK peptides 32-50 (residues 25-43) also show the same behavior as H-NOX peptides 7-12 and 21-26, suggesting that they are involved in the same binding interface. Mapping the Vc H-NOX/Vc HK binding interface in the presence of NO Next, the effect of NO on the H-NOX/HK interaction was examined. For this experiment, H-NOX was pre-treated with NO to form the H-NOX Fe(II)–NO complex before incubating with the HK. The heat map of H-NOX Fe(II)–NO upon HK binding showed significantly slower exchange (Δ%D 0), suggesting a global conformational change induced by NO-bound Vc H-NOX. c) Model of Vc H-NOX/Vc HK N-terminal domain interaction. Left: in the absence of NO, H-NOX interacts with the HK N-terminal domain primarily through H-NOX helices αA–C; right: in the presence of NO, the conformational change induced by NO binding to Vc H-NOX creates a new contact between the H-NOX αF helix and part of the helical region of the HK N-terminal.


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Biochemistry

Based on the HDX experiments, HK appears to be more sensitive to binding-induced solvent protection than Vc H-NOX. In other words, the extent of exchange is more suppressed for HK than H-NOX in the complex. Since the rate of deuterium exchange depends on both solvent protection and hydrogen-bonding with surrounding residues, it is possible that the H-NOX/HK complex formation causes a conformational change of the HK N-terminal domain to increase the homodimer interface or hydrogen-bonding within HK33. This could explain why no new surface on HK corresponding to H-NOX αF helix binding was found when NO was present (Figure 6a). This interface may be part of the HK N-terminal domain that was interacting more weakly with the H-NOX αF helix in the absence of NO (Figure 4b). Taken together, a model can be proposed for the protein/protein interaction between Vc H-NOX and Vc HK N-terminal region in response to NO (Figure 6c). When NO is absent, the N-terminal domain of Vc HK is only interacting with the helices αA-C regions of Vc H-NOX. When NO is present, the rotation of the H-NOX αF helix forms additional contacts within the HK N-terminal domain, which could then contribute the regulation of the kinase activity. Validating the HDX-MS-mapped binding interface by pulldown assay To confirm that areas of reduced H/D exchange on both proteins are a result of direct protein/protein interaction, a GST pulldown assay was used to validate the HNOX/HK interface mapped by HDX-MS. First, an alanine scan of surface-exposed charged residues on H-NOX was performed (Figure S8a,b). While modest binding disruption was observed for most of the alanine mutants, the D23A variant exhibited significant disruption of Vc HK binding. To further investigate the binding surface, a more intensive mutagenesis strategy was used that targeted aromatic residues and created charge reversal mutations on the H-NOX. This approach resulted in several mutants with reduced HK binding (Figure 7a,b; Figure S8c,d). When mapped onto the Vc H-NOX homology model, mutations with decreased affinity (Y6A, D11R, E15R, W22A, D23R) all faced one side of the αA and αB helices, while those with less binding disruption (S10E, F17A, F21A) pointed to the opposite side of those helices. Mutations on the loop between αA and αB (E34R and V36E) also disrupted kinase binding. These results are consistent with the HDX-MS experiments that showed the H-NOX αA and αB helices and part of the loop region are critical for HK N-terminal domain docking. Furthermore, mutations distant from helices αA-αC showed no significant change in HK binding (Figure 7a,b). Specifically, mutations located on αF helix (K93E, D100R, K101E, K105E, E106R, R109E, D113R, E120R) did not affect kinase binding, indicating that although this region exhibits NO-induced conformational change, it is not involved in direct recruitment of the kinase, which agrees with the proposed model. This result was further validated by a kinase assay with H-NOX Fe(II)-NO D23A and R109A mutants; both failed to inhibit kinase activity in an NO-dependent manner (Figure S9).


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Figure 7. Pulldown validation of Vc H-NOX/Vc HK binding interface. a) Pulldown assay of Vc H-NOX charge reversal variants in the absence of NO. Bands are quantified and normalized to HisGST Vc HK intensity. Variants with relative band intensity less than 75% are identified as residues directly involved in Vc HK binding. Assays were performed with two independent replicates and error bars represent one standard deviation. b) Pulldown results in a) mapped on the Vc H-NOX homology model. c) Pulldown assay of the HisGST Vc HK N-terminal domain alanine variants in the absence of NO. d) Pulldown results in c) mapped on the Vc HK N-terminal domain homology model and HNOX/HK N-terminal domain complex model in Figure 6c.


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Biochemistry

To further probe the HK binding surface, a combination of domain truncations and mutagenesis was utilized (Figure S10a–d). Only constructs containing the Nterminal domain were able to interact with the H-NOX as seen in the pulldown assay. In addition, mutations of charged residues in this region showed significant disruption of H-NOX binding, especially residues R9, R13, R15, R18 and E22, suggesting that they make critical contacts with H-NOX (Figure S10e,f; Figure 7c). Interestingly, the Vc HK N-terminal domain is predicted to be an α-helix with R9 and R13 facing one direction and R15, R18, E22 facing another direction (Figure 7d). If all five residues are necessary to make direct contact with the H-NOX, it is very likely that the helix bends between R13 and R15, further supporting the proposed model (Figure 6c, Figure 7d).

Figure 8. Proposed model for NO-induced Vc H-NOX inhibition of Vc HK kinase activity. a) In the absence of NO, Vc H-NOX interacts with the Vc HK N-terminal domain mainly through Vc H-NOX helices αA–C (binding interface highlighted in light blue) so the HK DHp and CA domains can still move freely and the ATP binding and phosphotransfer are unaffected. b) When NO is present, the conformational change induced by NO binding to HNOX creates a new contact between the H-NOX αF helix and potentially part of the HK Nterminal domain near the DHp (binding interface highlighted in dark blue). This new contact likely introduce more conformational changes in the HK DHp and CA domains that inhibited the kinase autophosphorylation by either prevent ATP binding to the CA domain or restrict the phosphotransfer from CA domain to DHp H71.


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Concluding remarks In this study, we successfully mapped the Vc H-NOX/Vc HK binding interface on both proteins by HDX-MS and subsequently validated key residues involved in this interaction. By comparing the binding interface in unliganded and NO-bound HNOX, a model for NO-dependent HK inhibition can be proposed (Figure 8). In the absence of NO, H-NOX and HK primarily anchored through the αA-C helices of the HNOX and a portion of the N-terminal domain of the HK. This allows the kinase DHp and CA domains to move freely and adopt a kinase-active conformation (Figure 8a). In the presence of NO, conformational changes in the H-NOX αF helix create an additional binding interface with the HK, likely through the C-terminal half of the Nterminal domain. This contact induces a further conformational change in the HK DHp and CA domains that renders the protein inactive by preventing ATP binding or phosphotransfer29. This could be achieved by either destabilizing the protein fold or altering the relative orientation of the two domains (Figure 8b). It is worth noting that there is no direct H-NOX binding to the HK phosphoacceptor histidine or CA domains, indicating that the mechanism of kinase inhibition is through allosteric regulation where the H-NOX induced conformational changes are propagated from the movement of the N-terminal domain to the DHp, rather than steric hindrance 29, 34-36. To summarize, this study provided structural insight that may be general for NOinduced H-NOX conformational changes and the interactions of H-NOX with the cognate sensor proteins. We have provided another example of the H-NOX distal alpha helices serving as the key interface for association with its sensor proteins14, 16. Additionally, we examined the details of kinase inhibition transduced by conformational changes in H-NOX and demonstrated that the rotation of the H-NOX αF helix is the critical step to regulate sensor protein activity, as predicted by previous crystallographic studies10, 27. Although structures of the H-NOX/HK complex are still needed to fully visualize the domain arrangements and conformational changes induced by NO binding, the current study validates the protein/protein interaction in solution and the model presented here furthers our understanding of the detailed mechanism of NO-mediated regulation in H-NOX/HK complexes under physiological conditions.


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Biochemistry

Supporting information Time dependence of Vc HK autophosphorylation inhibition by Vc H-NOX Fe(II)–NO (Figure S1); HDX-MS analysis of NO binding to Vc H-NOX (Figure S2); HDX-MS analysis of Vc HK binding to Vc H-NOX Fe(II) (Figure S3); HDX-MS analysis of Vc HNOX Fe(II) binding to Vc HK (Figure S4); HDX-MS analysis of Vc HK binding to Vc HNOX Fe(II)–NO (Figure S5); HDX-MS analysis of Vc H-NOX Fe(II)–NO binding to Vc HK (Figure S6); HDX-MS analysis of Vc HK at different Vc HK/Vc H-NOX ratios (Figure S7); Validating the Vc H-NOX binding interface by pulldown assay with HNOX mutants (Figure S8); Validating the Vc H-NOX binding interface by kinase assay (Figure S9); Validating the Vc HK binding interface by pulldown assay with HisGST Vc HK truncations and N-terminal alanine mutations (Figure S10). Abbreviations CA, catalytic domain; cGMP, guanosine 3¢,5¢-cyclic monophosphate; DEA, diethylamine; DHp, dimerization and histidine phosphotransfer domain; GST, glutathione-S-transferase; GTP, guanosine 5¢-triphosphate; H-NOX, Heme-Nitric oxide/Oxygen protein; HDX-MS, hydrogen/deuterium exchange mass spectrometry; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HK, histidine kinase; MBP, maltose binding protein; MCP, methyl-accepting chemotaxis protein; NO, nitric oxide; NTD, N-terminal domain; pGpG, diguanylic acid; RR, response regulator; SECMALS, size-exclusion chromatography multi-angle light scattering; sGC, soluble guanylate cyclase; TCEP, tris(2-carboxyethyl)phosphine; TEV, Tobacco Etch Virus protease; TFA, trifluoroacetic acid Author Contributions Y.G. and M.A.M. designed the research; Y.G. and M.M.C. performed the cloning, protein expression and purification. Y.G. performed the SEC-MALS, pulldown assays and kinase assays; Y.G. and A.T.I. performed the HDX-MS and data analysis. Y.G., A.T.I. and M.A.M. wrote the paper. Notes The authors declare no competing financial interest. Acknowledgements This work was supported by NIH (1S10OD020062-01 to A.T.I.). We would like to thank current and former members of the laboratory of Michael A. Marletta for frequent helpful discussions and invaluable insight, particularly Dr. Charles W. Hespen, Dr. Christopher M. Lemon, Benjamin G. Horst, Dr. Joel J. Bruegger and Dr. Minxi Rao. We would also like to thank Dr. Adam Offenbacher (Judith Klinman lab) for help with the HDX-MS experiment design/data analysis and Laura Nocka (John Kuriyan lab) for help with the SEC-MALS. All parallel expression vectors were kindly provided by Kevin H. Gardner’s lab at CUNY Advanced Science Research Center.


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References [1] Derbyshire, E. R., and Marletta, M. A. (2012) Structure and regulation of soluble guanylate cyclase, Annu Rev Biochem 81, 533-559. [2] Russwurm, M., and Koesling, D. (2004) NO activation of guanylyl cyclase, EMBO J 23, 4443-4450. [3] Zhao, Y., Brandish, P. E., Ballou, D. P., and Marletta, M. A. (1999) A molecular basis for nitric oxide sensing by soluble guanylate cyclase, Proc Natl Acad Sci U S A 96, 14753-14758. [4] Fernhoff, N. B., Derbyshire, E. R., and Marletta, M. A. (2009) A nitric oxide/cysteine interaction mediates the activation of soluble guanylate cyclase, Proc Natl Acad Sci U S A 106, 21602-21607. [5] Boon, E. M., Davis, J. H., Tran, R., Karow, D. S., Huang, S. H., Pan, D., Miazgowicz, M. M., Mathies, R. A., and Marletta, M. A. (2006) Nitric oxide binding to prokaryotic homologs of the soluble guanylate cyclase beta1 H-NOX domain, J. Biol. Chem. 281, 21892-21902. [6] Karow, D. S., Pan, D., Tran, R., Pellicena, P., Presley, A., Mathies, R. A., and Marletta, M. A. (2004) Spectroscopic characterization of the soluble guanylate cyclase-like heme domains from Vibrio cholerae and Thermoanaerobacter tengcongensis, Biochemistry 43, 10203-10211. [7] Pellicena, P., Karow, D. S., Boon, E. M., Marletta, M. A., and Kuriyan, J. (2004) Crystal structure of an oxygen-binding heme domain related to soluble guanylate cyclases, Proc Natl Acad Sci U S A 101, 12854-12859. [8] Plate, L., and Marletta, M. A. (2013) Nitric oxide-sensing H-NOX proteins govern bacterial communal behavior, Trends Biochem Sci 38, 566-575. [9] Iyer, L. M., Anantharaman, V., and Aravind, L. (2003) Ancient conserved domains shared by animal soluble guanylyl cyclases and bacterial signaling proteins, BMC Genomics 4, 5. [10] Hespen, C. W., Bruegger, J. J., Phillips-Piro, C. M., and Marletta, M. A. (2016) Structural and Functional Evidence Indicates Selective Oxygen Signaling in Caldanaerobacter subterraneus H-NOX, ACS Chem Biol 11, 2337-2346. [11] Imlay, J. A. (2003) Pathways of oxidative damage, Annu Rev Microbiol 57, 395418. [12] Ma, X., Sayed, N., Beuve, A., and van den Akker, F. (2007) NO and CO differentially activate soluble guanylyl cyclase via a heme pivot-bend mechanism, EMBO J 26, 578-588. [13] Price, M. S., Chao, L. Y., and Marletta, M. A. (2007) Shewanella oneidensis MR-1 H-NOX regulation of a histidine kinase by nitric oxide, Biochemistry 46, 13677-13683. [14] Lahiri, T., Luan, B., Raleigh, D. P., and Boon, E. M. (2014) A structural basis for the regulation of an H-NOX-associated cyclic-di-GMP synthase/phosphodiesterase enzyme by nitric oxide-bound H-NOX, Biochemistry 53, 2126-2135. [15] Plate, L., and Marletta, M. A. (2012) Nitric oxide modulates bacterial biofilm formation through a multicomponent cyclic-di-GMP signaling network, Mol Cell 46, 449-460.


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[16] Rao, M., Herzik, M. A., Iavarone, A. T., and Marletta, M. A. (2017) Nitric OxideInduced Conformational Changes Govern H-NOX and Histidine Kinase Interaction and Regulation in Shewanella oneidensis, Biochemistry 56, 12741284. [17] Janoff, E. N., Hayakawa, H., Taylor, D. N., Fasching, C. E., Kenner, J. R., Jaimes, E., and Raij, L. (1997) Nitric oxide production during Vibrio cholerae infection, Am J Physiol 273, G1160-1167. [18] Nisbett, L. M., and Boon, E. M. (2016) Nitric Oxide Regulation of H-NOX Signaling Pathways in Bacteria, Biochemistry 55, 4873-4884. [19] Mukhopadyay, R., Sudasinghe, N., Schaub, T., and Yukl, E. T. (2016) Hemeindependent Redox Sensing by the Heme-Nitric Oxide/Oxygen-binding Protein (H-NOX) from Vibrio cholerae, J Biol Chem 291, 17547-17556. [20] Wu, G., Liu, W., Berka, V., and Tsai, A. L. (2013) The selectivity of Vibrio cholerae H-NOX for gaseous ligands follows the "sliding scale rule" hypothesis. Ligand interactions with both ferrous and ferric Vc H-NOX, Biochemistry 52, 94329446. [21] Woodward, J. J., Martin, N. I., and Marletta, M. A. (2007) An Escherichia coli expression-based method for heme substitution, Nat Methods 4, 43-45. [22] Guo, Y., Suess, D. L. M., Herzik, M. A., Jr., Iavarone, A. T., Britt, R. D., and Marletta, M. A. (2017) Regulation of nitric oxide signaling by formation of a distal receptor-ligand complex, Nat Chem Biol 13, 1216-1221. [23] Sheffield, P., Garrard, S., and Derewenda, Z. (1999) Overcoming expression and purification problems of RhoGDI using a family of "parallel" expression vectors, Protein Expr Purif 15, 34-39. [24] Biasini, M., Bienert, S., Waterhouse, A., Arnold, K., Studer, G., Schmidt, T., Kiefer, F., Gallo Cassarino, T., Bertoni, M., Bordoli, L., and Schwede, T. (2014) SWISSMODEL: modelling protein tertiary and quaternary structure using evolutionary information, Nucleic Acids Res 42, W252-258. [25] Bordoli, L., Kiefer, F., Arnold, K., Benkert, P., Battey, J., and Schwede, T. (2009) Protein structure homology modeling using SWISS-MODEL workspace, Nat Protoc 4, 1-13. [26] Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling, Bioinformatics 22, 195-201. [27] Herzik, M. A., Jr., Jonnalagadda, R., Kuriyan, J., and Marletta, M. A. (2014) Structural insights into the role of iron-histidine bond cleavage in nitric oxide-induced activation of H-NOX gas sensor proteins, Proc Natl Acad Sci U S A 111, E4156-4164. [28] Glover, J. N., and Harrison, S. C. (1995) Crystal structure of the heterodimeric bZIP transcription factor c-Fos-c-Jun bound to DNA, Nature 373, 257-261. [29] Diensthuber, R. P., Bommer, M., Gleichmann, T., and Moglich, A. (2013) Fulllength structure of a sensor histidine kinase pinpoints coaxial coiled coils as signal transducers and modulators, Structure 21, 1127-1136. [30] Pascal, B. D., Willis, S., Lauer, J. L., Landgraf, R. R., West, G. M., Marciano, D., Novick, S., Goswami, D., Chalmers, M. J., and Griffin, P. R. (2012) HDX


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workbench: software for the analysis of H/D exchange MS data, J Am Soc Mass Spectrom 23, 1512-1521. [31] Schneider, C. A., Rasband, W. S., and Eliceiri, K. W. (2012) NIH Image to ImageJ: 25 years of image analysis, Nat Methods 9, 671-675. [32] Resing, K. A., Hoofnagle, A. N., and Ahn, N. G. (1999) Modeling deuterium exchange behavior of ERK2 using pepsin mapping to probe secondary structure, J Am Soc Mass Spectrom 10, 685-702. [33] Hoofnagle, A. N., Resing, K. A., and Ahn, N. G. (2003) Protein analysis by hydrogen exchange mass spectrometry, Annu Rev Biophys Biomol Struct 32, 1-25. [34] Wang, L. C., Morgan, L. K., Godakumbura, P., Kenney, L. J., and Anand, G. S. (2012) The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm, EMBO J 31, 2648-2659. [35] Moglich, A., Ayers, R. A., and Moffat, K. (2009) Design and signaling mechanism of light-regulated histidine kinases, J Mol Biol 385, 1433-1444. [36] Rivera-Cancel, G., Ko, W. H., Tomchick, D. R., Correa, F., and Gardner, K. H. (2014) Full-length structure of a monomeric histidine kinase reveals basis for sensory regulation, Proc Natl Acad Sci U S A 111, 17839-17844.


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