Tyrosine Nitration on Calmodulin Enhances calcium-dependent association and activation of Nitric Oxide Synthase

Production of reactive oxygen species due to dysregulated endothelial nitric oxide synthase on calcium binding by calmodulin and on binding and activation of eNOS. We found that nitroTyr-calmodulin retains affinity for eNOS under resting physiological calcium concentrations. Results from in vitro eNOS assays with calmodulin nitrated at Tyr-99 revealed that this nitration reduces nitric oxide production and increases eNOS decoupling compared with wild-type calmodulin. In contrast, calmodulin nitrated at Tyr-138 produced more nitric oxide and did so more efficiently than wild-type calmodulin. These results indicate that the nitroTyr posttranslational modification, like tyrosine phosphorylation, can impact calmodulin sensitivity for calcium and reveal Tyr site– specific gain-or loss-of-functions for calmodulin-induced eNOS activation.


Introduction
Fundamental biological processes like cell proliferation, gene transcription, cell death, exocytosis, and metabolism are predicated on the tight regulation of cytosolic calcium (Ca 2+ ) concentration (1). At the heart of Ca 2+ regulation is the Ca 2+ sensing protein, calmodulin (CaM), which transduces intracellular Ca 2+ signals into biological responses. CaM accomplishes this by serving as a Ca 2+ binding regulatory subunit of a wide array of enzymes, structural proteins, and membrane transporters. Cellular CaM fluctuates between its Ca 2+ bound form (Ca 2+ -CaM) and Ca 2+ and target protein bound forms (Ca 2+ -CaM-target) depending on combination of intracellular Ca 2+ levels and target affinity. Cytosolic Ca 2+ levels are tightly regulated between ~0.1 µM under resting conditions to ~10 µM after stimulation (2). CaM is an abundant protein with intracellular concentrations up to 10 µM, however due to the collective concentration of its many target proteins, CaM is the limiting agent in Ca 2+ sensing (3,4).
CaM target proteins in high concentration or that bind CaM with the highest affinity will scavenge the CaM from the cell. At maximal intracellular calcium levels, 25% of the Ca 2+ -CaM pool in endothelial cells is associated with endothelial nitric oxide synthase (eNOS) (3). This amount of CaM bound by tight association with eNOS removes sufficient CaM from the intracellular pool to affect other target proteins as seen with the intracellular calcium regulatory channel, plasma membrane Ca 2+ -ATPase (3). Posttranslational modifications (PTMs) on CaM serve as an additional level of control over CaM-target affinity and CaM activity necessary for the intracellular Ca 2+ regulatory balance. CaM has two tyrosines, Tyr99 and Tyr138, which are phosphorylated at different stoichiometries to regulate target protein association (3,6). Tyrosine phosphorylation of CaM has been shown to impact Ca 2+ -dependent CaM interaction with nitric oxide synthase (NOS). PhosphoCaM-99 binds 4 times more tightly to neuronal NOS as compared to WT-CaM (4).
CaM is also susceptible to oxidative post-translational modifications (ox-PTMs) by reactive oxygen species (ROS) and reactive nitrogen and oxygen species (RNS), such as hydrogen peroxide (H2O2), nitric oxide (NO), and peroxynitrite (ONOO -) (5)(6)(7)(8). Proteomic analysis has shown that ROS and RNS induce a variety of ox-PTMs on CaM in vivo, resulting in an accumulation of significant amounts of tyrosine nitration, and methionine sulfoxidation (9)(10)(11)(12). Previous studies have indicated that nitration of CaM at tyrosine 99 is a biomarker of oxidative stress and that nitration of CaM at tyrosine 138 is subject to a denitrase activity in activated macrophages (5,11). Up to 30% of the cellular CaM pool is nitrated on critical regulatory tyrosine residues following macrophage activation (5), however, the effect of tyrosine nitration on CaM function has not been determined. However, given the impact of phosphorylation of these tyrosines on the CaM-NOS interaction, it's perhaps not surprising to think that CaM tyrosine nitration will alter CaM-eNOS affinity.
To evaluate the impact of ox-PTMs on CaM, previous work used standard site-directed mutagenesis with unreactive canonical amino acids to control sites of chemical oxidation. This approach, while the best available at the time, is problematic because altering amino acids to prevent ox-PTM formation can have unknown effects at these regulatory hot-spots (11). Genetic code expansion (GCE) provides the means to assess the effects of site-specific protein tyrosine nitration through cotranslational installation of nitroTyr into the protein of interest. This methodology has been used recently to show that tyrosine nitration can cause a toxic gain-of-function for key regulatory sites in heat shock protein 90 (Hsp90) and apolipoprotein A-I (apoAI) as well as a loss of function in manganese superoxide dismutase (Mn-SOD) (13)(14)(15). The abundance of tyrosine nitration on CaM led us to characterize the effect this ox-PTM has on altering CaM function with site-specifically modified nitroTyr-containing CaM generated with GCE. We focused on CaM target eNOS because it utilizes a significant fraction of the cellular CaM pool and because of the regulatory role of tyrosine phosphorylation in the system. ENOS is a multi-domain enzyme that catalyzes the conversion of L-arginine to the biological signaling molecule nitric oxide to regulate vascular tone and angiogenesis (16).
In addition, oxidative stress has been shown to alter nitric oxide levels and compromise the regulation of vascular tone and angiogenesis (12,18).
Using homogeneously nitrated CaM, we found that tyrosine nitration increases the affinity of CaM for it's key target protein, eNOS, at reduced calcium levels, and interestingly, site-specific tyrosine nitration also increases its ability to produce the product, NO. This study provides the first evidence that tyrosine nitration of CaM can provide a gain-offunction to the Ca 2+ -dependent activation of eNOS signaling.
We sought to characterize the functional effects of site-specifically nitrating the tyrosine residues on CaM due to nitroTyr-CaM abundance in cells. We installed nitroTyr co-translationally into CaM at positions 99 and 138 using GCE to generate homogeneous nitroTyr-CaM (Fig. 1). When nitroTyr was withheld from the media, no full-length protein was purified, indicating the fidelity of the engineered aaRS/tRNA pair for nitroTyr (Fig.  1B). The fidelity of site-specific nitroTyr incorporation was verified by mass spectrometry, with a mass increase of 44 Da corresponding to the addition of a single nitro group (Fig. 1D). The presence of nitroTyr was further confirmed by anti-nitroTyr Western blot (Fig. 1C). These cumulative results validate the specific incorporation of nitroTyr into CaM.

NitroTyr-CaM Exhibits a Gain-of-Function Interaction with Target Protein eNOS
To determine if nitration of CaM changed its Ca 2+ -dependent ability to bind target proteins we supplemented EA.hy926 human endothelial cell lysate with the sitespecific nitroTyr-CaM-V5 tag at 20% of the endogenous CaM concentration. Immunoprecipitation of CaM co-precipitated a clearly evident protein with an apparent molecular weight of 130 kDa, consistent with the CaM target eNOS (Fig. 2). As eNOS was co-immunoprecipitated by both WT-and nitroTyr-CaM-V5 tag, this indicates that nitroTyr-CaM-V5 interacts with eNOS in the presence of other intracellular targets of CaM.
To determine the extent of the impact of nitration of CaM on its Ca 2+ -dependent ability to bind eNOS we employed the use of biolayer interferometry (BLI), outlined in figure 3. This method allows for uniform attachment of a target protein-binding region to a BLI optical biosensor tip and measurement of CaM binding in solutions containing 20-225 nM buffered Ca 2+ . 43 To determine if CaM nitration alters its affinity for eNOS we used the synthesized 20 amino acid peptide CaM binding domain of eNOS with an N-terminal PEGbiotin to aid in solubility and allow for defined surface attachment to BLI streptavidin biosensor tips. The association and dissociation of CaM proteins to immobilized eNOS peptide was fit to a 1:1 interaction model which provides the Kd under different Ca 2+ concentrations (Table 1, Fig. 3) (20,21).
Consistent with values reported in the literature, WT-CaM binding of eNOS peptide affinity ranges from ~1 nM to undetectable levels as [Ca 2+ ]free is decreased from saturating levels (2 mM) to physiologically resting levels (20-50 nM) (17). Both nitroTyr-CaM species exhibit similar eNOS-peptide binding affinity to WT-CaM at saturating calcium levels. Strikingly, both of the nitroTyr-CaM species retain ~5 nM affinity at physiologically resting [Ca 2+ ]free, compared to undetectable affinity for WT-CaM. Due to the retention of high affinity for eNOS at resting [Ca 2+ ]free, nitroTyr-CaM has the potential to constitutively activate eNOS in the absence of calcium signal resulting in a gain-of-function alteration regulating eNOS activity.

NitroTyr-CaM Exhibits a Gain-of-Function Activation of eNOS Function
Based on the BLI data, we can conclude that nitration of CaM leads to CaM-eNOS association at resting intracellular calcium concentrations, however this change in affinity only has a regulatory gain-of-function if these nitration sites on CaM do not abolish eNOS enzyme activity. Electron transfer between eNOS domains is dependent on the reversible binding of CaM, which is governed through changes in the intracellular Ca 2+ concentration. To determine if the increased binding affinity of nitroTyr-CaM altered eNOS activity we measured the steady-state NO synthesis activities of WT-eNOS in presence of WT-CaM and nitroTyr-CaMs ( Table 2). The coincident rates of NADPH oxidation during the assays were also measured to determine the efficiency of NO production. The cytochrome c reduction assay also provides a means to assess correct electron transfer through the enzyme. Generally, these assays are conducted under saturating calcium levels (2 mM free Ca 2+ concentration). At these Ca 2+ concentrations, all CaM species bind eNOS with the same affinity (Table 1), so any differences in NO production seen are independent of CaM-eNOS affinity. It is not feasible to perform eNOS assays at very low Ca 2+ concentrations due to slow eNOS turnover and detection limits of the oxyhemoglobin, NADPH, and cytochrome c assays. At saturating Ca 2+ levels, the eNOS was ~1.3 times more active with nitroTyr-CaM-138 than eNOS with WT-CaM. NitroTyr-CaM-99 had an inhibitory effect and lowered NO synthesis by ~40 %. We also compared how these CaMs impact the NADPH oxidation rates of WT-eNOS during NO synthesis from L-Arg. In general, the rate of NADPH oxidation followed the rate of NO synthesis, consistent with the coupling of these processes. Remarkably, the coupling in WT-eNOS with nitroTyr-CaM-138 (3.0 NADPH per NO) was more efficient than in WT-eNOS with WT-CaM (4.4 NADPH per NO). This means that that the eNOS works more efficiently with nitroTyr-CaM-138, and enables greater NO synthesis without an increased production of other reactive oxygen species.
The steady-state cytochrome c reductase activity is a useful way to measure the electron flux passing through the NOS FMN subdomain. Due to the closed conformation of the NOS reductase domain cytochrome c activity is suppressed in the absence of CaM and cytochrome c activity increases in the presence of CaM. A typical 5-fold increase in cytochrome c activity was induced by the binding of WT-CaM to WT-eNOS in our assays (18). We found that nitroTyr-CaM-138 has a 10 % increase in activity as compared to WT-CaM and that nitroTyr-CaM-99 has a 10 % decrease in activity as compared to WT-CaM. Cytochrome c reductase activity data also indicate that the eNOS works more efficiently with nitroTyr-CaM-138 than WT-CaM. When supplemented with either of the nitroTyr-CaM species as opposed to WT-CaM, eNOS retained more activity at 225 nM [Ca 2+ ]free (Table 3). This indicates that the gain-of-function seen with nitroTyr-CaM-eNOS binding is conserved with the full-length protein.

NitroTyr-CaM interacts with and stimulates the function of eNOS in eNOS-expressing HEK293 cell lysate
To recapitulate the effects seen in vitro with the pure CaM-eNOS system in a system more closely resembling the intracellular medium, we used lysate from eNOS-producing HEK293 cells, allowing us to still control both the concentration of supplemented CaM and [Ca 2+ ]free and verified that all CaM forms were able to stimulate eNOS activity in HEK293-eNOS lysate. As expected for eNOS we see a calcium concentration- (Fig. 4) and time-(data not shown) dependent increase in activity. This activity is responsive to the NOS inhibitor L-NAME (data not shown) indicating that NO synthesis is a result of eNOS activity. Due to the presence of endogenous CaM there was a significant eNOS activity without the addition of exogenous CaM (Fig. 4). As predicted from the increased affinity of nitroTyr-CaM-138 for eNOS at low calcium concentrations, the eNOS activity was stimulated to a significantly larger extent in lysate supplemented with nitroTyr-CaM-138 over WT-CaM (Fig. 4), particularly at intermediate calcium levels (225-750 nM [Ca 2+ ]free). Specifically, we see a 60-70% increase in NO production by nitroTyr-CaM-138 over WT-CaM at these intermediate calcium levels.

Nitration of CaM Does Not Impair Calcium Binding
It has been shown that CaM PTMs regulate target affinity and activity by altering interactions with specific targets but another potential mechanism would be for the PTM to alter CaMs affinity for Ca 2+ . Dansylated CaM (dansyl-CaM) fluorescence has also been used for monitoring conformational changes in CaM as a result of interactions with Ca 2+ (19). To analyze the Ca 2+ induced structural changes of the different dansyl-CaM species, we performed Ca 2+ fluorescence titration experiments (Fig. 5). Fitting the data (equation 2, materials and methods) gives the EC50(Ca 2+ ), the free calcium concentration at which the dansyl-CaM is half saturated with Ca 2+ , and n, the Hill cooperativity constant ( Table 4). The EC50(Ca 2+ ) values for all CaM forms were approximately 0.3 µM [Ca 2+ ]free. This unaltered Ca 2+ affinity for the WT-and nitroTyr-CaMs is in good agreement with previously published dansyl-CaM Ca 2+ affinity data (27). The Ca 2+ fluorescence titration did show a difference for the Hill cooperativity coefficient for the WT-as compared to the nitroTyr-CaMs (Table 4). This indicates that there is slightly lower cooperativity for the nitroTyr-CaMs as compared to the WT-CaM. The fluorescence titration data confirms that tyrosine nitration on CaM does not independently change its affinity for Ca 2+ , which indicates the increased nitroTyr-CaM-eNOS affinity at low Ca 2+ concentrations is due to cooperative binding of both target eNOS and Ca 2+ binding to nitrated CaM.

Discussion
Previous work has shown that eNOS is almost exclusively CaM-free under resting conditions but is half-bound at roughly a quarter of maximum intracellular [Ca 2+ ]free (50% saturation of the eNOS peptide at a [Ca 2+ ]free of 228 nM) (27). While unmodified CaM is inactive under resting [Ca 2+ ]free, tyrosine phosphorylation of CaM increases binding affinity for NOS compared to unphosphorylated CaM (4). The sites of phosphotyrosine regulation on CaM are also shared by tyrosine nitration, which makes it important to understand how this ox-PTM alters CaM signaling. CaM is maintained at low basal levels of tyrosine phosphorylation and nitration. In response to insulin signaling or infection by Rous sarcoma virus, the amount of phosphotyrosine increases up to 50% and nitrotyrosine levels increase up to 30% due to oxidative stress and immune responses (5,20). The balance of CaM phosphorylation and nitration further broadens the pool of regulatory components that control eNOS function (5,(21)(22)(23).
Our results show nitroTyr-CaM-138 remains bound even at 20 nM [Ca 2+ ]free, indicating that eNOS in the presence of nitroTyr-CaM-138 is active under resting conditions, analogous to what was seen for phosphoCaM-99. Both of the nitroTyr-CaM species bind the same as the WT-CaM with identical sub-nanomolar affinities under saturating [Ca 2+ ]free, however they exhibit a slight decrease in affinity for the eNOS peptide at the resting [Ca 2+ ]free of 50 nM. Previously, phosphorylation of eNOS at S1179 by Akt or PKA has shown a gain-of-function by allowing CaM to bind at lower [Ca 2+ ]free levels, effectively lowering the calcium signal required for eNOS activation (24). In the same vein, nitration of CaM at Tyr 138 provides a similar but newly uncovered mechanism for enhancing CaM interactions and thereby activating eNOS independent of an increase in the [Ca 2+ ]free level (Fig. 6). Tyr138 serves as a structural coupler between the N-terminal domain and the central linker of CaM through hydrogen bonding with Glu82 ( Fig. 6) (25), potentially explaining the alteration in eNOS association seen following nitration at this site. While phosphorylation of CaM has been shown to alter the affinity of CaM for eNOS, it also clear that phosphorylation of CaM or eNOS also influence the activity of eNOS in a manner independent from CaM-eNOS affinity. Based on this, we expect that tyrosine nitration of CaM will impact eNOS function in addition to the changes to affinity.
In order to study the influence of CaM nitration on the catalytic activity of full-length WT-eNOS, we monitored NO production, NADPH oxidation, and cytochrome c reduction.
Previous work with the phosphomimetic CaM mutation Y99E showed a 40% decrease in NO production, while the control mutation Y99Q only showed a 20% decrease in NO production (23). At 2 mM [Ca 2+ ]free, similar to the Y99E mutation, nitroTyr-CaM-99 resulted in a 40% decrease in NO production and was less efficient with a 10% decrease in NADPH to NO ratio as compared to WT-CaM. Strikingly nitroTyr-CaM-138 was able to support more NO production, and more efficiently, than WT-CaM, with a 33% increase in NO output and a 62% efficiency increase. This increase in output and efficiency is comparable to what was seen for eNOS regulation by the phosphomimetic mutations S1179D or S617D (18,26).
As we do not expect the affinity of nitroTyr-CaM for eNOS to be altered at saturating [Ca 2+ ]free as compared to WT-CaM we may be able to ascribe the change in eNOS function to a shift in eNOS domain dynamics (16) Because CaM regulates many proteins, the nitroTyr-CaM pool may impact target proteins other than eNOS. Regulation of eNOS is also multifaceted and is also dependent on interactions with proteins other than CaM and subcellular localization. In order to approach this problem, we first assessed eNOS-nitroTyr-CaM association in HEK293-eNOS and EA.hy926 human endothelial cell lysate. We saw that both nitroTyr-CaM species when supplemented to cell lysate at 20% of the total CaM pool, interacted with eNOS via immunoprecipitation of eNOS via the V5-tag on CaM. Assessing eNOS function, we saw that supplementation with nitroTyr-CaM, particularly at site 138, led to significantly more eNOS activity at intermediate calcium levels representing a gain-of-function even in the presence of native CaM, other CaM target proteins, and other eNOS regulatory interactions. Under this condition, the nitroTyr-CaM-138 does stimulate 60-70% more eNOS activity in cell lysate than does WT-CaM, consistent with a gain-of-function.

Conclusion
Here we sought to explore whether the nitroTyr PTM can alter the function of regulatory protein (27), in our case CaM. The central role CaM plays in regulating calcium signaling, abundant nitration of it's tyrosine residues, and the potential presence of a cellular denitrase for nitrated CaM (5) make it an ideal candidate for assessing the impact of tyrosine nitration. Since nitroTyr can be incorporated site-specifically using GCE, it provides excellent opportunity to evaluate if it can modulate protein function. Our results show that tyrosine nitration of CaM at sites 99 and 138 increase binding and activation of eNOS at lower Ca 2+ concentrations. Most strikingly, CaM nitrated at site 138 binds eNOS even under resting physiological conditions, indicating that while only a subset of cellular CaM will be nitrated on tyrosine 138 at any given time, due to its gain-of-function this subpopulation will activate eNOS at reduced [Ca 2+ ]. Proper regulation of eNOS is important for healthy vascular function, and tyrosine nitration enhances CaM calcium sensitivity and activation of eNOS. CaM binding to NOS activates NOS by relieving the suppression of the electron transfer process. Under conditions of limited arginine substrate or improper coupling of N-terminal oxygenase domain and C-terminal reductase domain NOS has also been observed to produce superoxide through electron transfer to molecular oxygen (28). As eNOS produces both superoxide and nitric oxide, which can react to form peroxynitrite (8,29), the nitrating agent of CaM, tyrosine nitration may serve as a positive feedback mechanism to stimulate eNOS function. Based on the efficiency of nitric oxide production, we would predict from these results that nitration of CaM at Tyr99 will lead to increased eNOS decoupling and therefore tyrosine nitration with lower NO bioavailability, while nitration at Tyr138 will decrease uncoupling and the level of tyrosine nitration, but increase the overall amount of bioavailable NO.

Immunoblotting
Western blot samples were separated on 15% SDS-PAGE gels, transferred to PVDF membrane, blocked with 5% nonfat milk in TBST, and probed with antinitrotyrosine (1:500) primary antibody, rocking for 16 hours at room temperature. After rinsing three times with TBST, the membranes were than incubated with Li-Cor IRDye 800CW Goat anti-Rabbit IgG (1:10,000) secondary antibody, rocking for 1 hour at room temperature, and washed three times for 5 minutes in TBST. The membrane was then scanned using a Li-Cor Odyssey 9120 Imaging System.

Recombinant expression of homogenous sitespecifically modified nitroCaM
The human CaM sequence was codon optimized for expression in E. coli (GenScript USA) and cloned into a pBad vector to include a C-terminal 6xhis affinity tag (Invitrogen). The protein was expressed via DH10B E. coli cells in autoinduction media in the presence of 100 µg/mL ampicillin for 24 hrs, at 37 °C, shaking at 250 rpm. Cells were pelleted at 5500 rcf then resuspended in approximately 10 mL binding/wash buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.4) at 4 °C and lysed once with a Microfluidics M-110P microfluidizer set at 18,000 psi. Cell debris was pelleted in Oakridge tubes at 20,900 rcf for 25 min at 4 °C. Approximately 75 mL of supernatant was passed through an Acrodisc 32 mm syringe filter fitted with a 0.45 um Supor membrane. The supernatant was loaded onto a 5 mL HisTrap NiNTA column at 1 ml/min, washed with 20 mL wash buffer, and eluted with 0-100%, 30 mL linear gradient of elution buffer (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.4) using an Amersham Pharmacia Biotech AKTA explorer. Peak elutions were between 30-40% elution buffer, corresponding to 150-200 mM imidazole. The pure protein fractions then had CaCl2 added to a concentration of 5 mM and loaded on a 5 mL HiTrap phenyl sepharose column, washed with 20 mL of wash buffer (50 mM Tris-HCl, 1 mM CaCl2, 500 mM NaCl, pH 7.5), and eluted with 0-100%, 30 mL linear gradient elution buffer (50 mM Tris-HCl, 1 mM EDTA, pH 7.5). Pure peak fractions determined via SDS-PAGE analysis were pooled and dialyzed for 4 hrs into storage buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.5) twice, fresh storage buffer was added for overnight dialysis. The purified CaM was concentrated via centricon tubes (3 kDa MWCO) to a concentration greater than 400 µM. All CaM used for co-IPs, eNOS function assays, BLI measurements, and calcium binding assays was prepared in this manner and used in this buffer unless noted elsewhere in the experimental procedures.
Expression and purification of CaM containing nitroTyr E. coli DH10B was transformed with pBad-CaM-(99 or 138 TAG) and pDule-nitroTyr-5B (Addgene Plasmid #85498) (30) The orthogonal aminoacyl-tRNA synthetase and cognate amber suppressing tRNA for incorporation of nitroTyr are expressed from the plasmid pDule containing the aforementioned synthetase/tRNA pair, a p15 origin, and a tetracycline resistance marker. The pBad-CaM plasmids contain CaM with an amber stop codon at the site of interest (99 or 138) to direct incorporation of nitroTyr. Similar to WT-CaM, expression of nitroTyr-containing CaM was in autoinduction media (31) containing 100 µg/mL ampicillin, 25 µg/mL tetracycline, and 1 mM nitroTyr. Purification of nitroTyr-containing CaM was identical to purification of WT-CaM. Yields of ~300 mg (L of culture) -1 were obtained for nitroTyr-CaM-99 and nitroTyr-CaM-138, compared to that of ~375 mg (L of culture) -1 for WT-CaM expression (31).

Mass Spectromectry
Purified CaM samples were diluted to a concentration of 10 µM, desalted on Millipore C4 zip tips, and analyzed using an FT LTQ mass spectrometer at the Oregon State University Mass Spectrometry Facility. Samples included WT-CaM and nitroTyr-containing CaM. Spectra were collected using Tune-Plus (Thermo, v. 2.2) page of Xcalibur (Thermo, v. 2.0.5) using parameters described in (32). Spectra were averaged over the three minutes CaM eluted from the ZipTip using the Qual Browser (Thermo, v. 2.0) The data were exported as text files containing two columns of m/z versus intensity to be analyzed by custom programs written in MatLab (32). The raw and deconvoluted mass spectrometry data deposited online.

Calmodulin dansylation and fluorescence measurements
Dansyl-CaM was prepared as previously described (23). CaM (1 mg/mL) was buffer exchanged into 10 mM NaHCO3 and 1 mM EDTA (pH 10.0) at 4 °C. Thirty µL of 6 mM dansyl chloride (1.5 mol/mol of CaM) in acetone was added to 2 mL of CaM, while it was being stirred. After incubation for 12 h at 4 °C, the mixture was buffer exchanged into fluorescence buffer. Labeling yields were determined from absorbance spectra using an ε320 of 3400 M −1 cm −1 and were compared to actual protein concentrations determined using the Bradford method with wild-type CaM used as the protein standard.
Fluorescence emission spectra were recorded using a PTI (London, ON) QuantaMaster spectrofluorimeter. Fluorescence measurements were taken on 50 µL samples consisting of dansyl-CaM (2 µM) in 30 mM MOPS, 100 mM KCl, and 10 mM EGTA (pH 7.2) with an increasing concentration of free Ca 2+ . The free Ca 2+ concentration was controlled using the suggested protocol from the calcium calibration buffer kit from Invitrogen. The excitation wavelength for all of the dansyl-CaMs was set to 340 nm, and emission was monitored between 400 and 600 nm. Slit widths were set at 2 nm for excitation and 1 nm for emission. The relative fluorescence was calculated with the equation: (1) where F is the measured intensity, Fmax is the maximal intensity, and F0 is the intensity without added Ca 2+ . The relative fluorescence was calculated using the fluorescence emission intensity at 475 nm. The Ca 2+ sensitivities of the different dansyl-CaMs we determined as the EC50(Ca 2+ ) values, which were derived from fits of the relative fluorescence intensity increase upon addition of Ca 2+ using the equation: Relative Fluorescence = [23 45 where relative fluorescence is obtained from equation 1; n is the Hill coefficient.

Octet Red96 Biolayer interferometry measurements
All BLI measurements were made on a fortéBIO (Menlo Park, CA) Octet Red96 system using streptavidin ('SA') sensors. Assays were performed in 96-well microplates at 37 °C. All sample volumes were 200 µL. The eNOS peptide was purchased from Genscript (Piscataway, NJ) homogeneously biotinylated at the N-terminus. Tips were loaded with three different concentrations of biotin-eNOS peptide (33). After loading biotinylated eNOS peptide onto SA sensors, a baseline was established in buffer composed of 30 mM MOPS, 100 mM KCl (pH 7.2), and varying free calcium concentration. Free calcium was controlled by mixing two buffers containing 10 mM EGTA and 10 mM Ca 2+ -EGTA in varying ratios. Free calcium concentration was calculated either from the ThermoFisher Scientific Calcium Calibration Kit #1 instructions or the Maxchelator program (34). Association with the analyte CaM was then carried out in the same buffer for 90 seconds at CaM concentrations of 180 nM, 60 nM, and 20 nM. Dissociation was subsequently measured in buffer without CaM over 1200 seconds. CaM used in the BLI experiments was unaltered following the purification method outlined above.

Steady-state eNOS assays
NO synthesis and NADPH oxidation rates were determined using the oxyhemoglobin assay. The NO synthesis activity was determined by the conversion of oxyhemoglobin to methemoglobin using an extinction coefficient of 38 mM -1 cm -1 at 401 nm. The NADPH oxidation rates were determined following the absorbance at 340 nm, using an extinction coefficient of 6.2 mM -1 cm -1 . Reaction mixtures (total volume 400 µl) contained 0.1 -0.2 µM bovine eNOS, 0.3 mM dithiotreitol, 4 µM FAD, 4 µM FMN, 10 µM H4B, 2 mM L-Arg, 0.1 mg/ml bovine serum albumin, 2 mM CaCl2, 0.2 mM EDTA, 2 -5 µM CaM (WT or nitroTyr depending on the experiment), 100 units/ml catalase, 60 units/ml superoxide dismutase, 5 µM oxyhemoglobin and 150 mM NaCl in 40 mM EPPS buffer, pH 7.6. The reaction was initiated by adding NADPH to a final concentration of 250 µM. In the assays without CaM, CaCl2 was omitted and EDTA concentration was increased to 0.45 mM. Cytochrome c reductase activity was determined by following the absorbance change for the reduction of cytochrome c by eNOS at 550 nm using an extinction coefficient of 21 mM -1 cm -1 . Reaction mixtures (total volume 400 µl) contained ≤ 0.01 µM eNOS, 25 µM FAD, 25 µM FMN, 0.1 mg/ml bovine serum albumin, 2 mM CaCl2, 0.2 mM EDTA, 1.0 -4.0 µM CaM (WT or nitroTyr depending on the experiment), 100 units/ml catalase, 40 units/ml superoxide dismutase, 65 µM cytochrome c and 150 mM NaCl in 40 mM EPPS buffer, pH 7.6. The reaction was initiated by adding NADPH to a final concentration of 250 µM. In the assays without CaM, neither CaM nor CaCl2 were added and EDTA concentration was 0.45 mM. All steady-state assays were carried out at 25 °C using a Hitachi 3110 spectrophotometer. Reported values are means ± SD of three or more determinations.

Production of the stable HEK293-eNOS line
The bovine eNOS sequence in a pcDNA3 vector and empty pcDNA3 vector were transfected into HEK293 cells (ATCC) using lipofectamine 2000 (Thermo Fischer Scientific) according to the manufacturer's protocol. Stable HEK293 cell populations were selected for using G418 at 400 µg/mL. Expression of eNOS was confirmed by western blot using pan-NOS antibody (Cell Signaling Technology).

Immunoprecipitation of V5-tagged WT-CaM and NitroTyr-CaM from HEK293-eNOS cells
ENOS was co-immunoprecipitated from cells (either HEK-eNOS expressing cells or EA.hy926 human endothelial cells) prepared in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1% Triton X-100, and 1x protease inhibitor cocktail). Recombinant WT-or nitroTyr-CaM-V5 tag was supplemented into the lysate at 20% of the endogenous CaM concentration in the lysate along with 5 mM CaCl2. The lysate with recombinant CaM was incubated at 4°C overnight with 2 µl mouse anti-V5 antibody (Invitrogen). The mixture was further incubated with 20 µl of protein A/G magnetic beads for 6 hours at 4°C. The magnetic beads were washed four times with lysis buffer containing 5 mM CaCl2, resuspended in 40 µl of Laemmli buffer, and incubated at 55 °C for 10 minutes. Western blot samples were separated on 4-22% gradient SDS-PAGE gels, transferred to PVDF membrane, blocked with 5% nonfat milk in TBST, and probed with antiNOS (Pan) (1:1000) or antiV5 (1:5000) primary antibodies rocking for 16 hours at room temperature. After rinsing three times with TBST, the membranes were than incubated with Li-Cor IRDye 800CW Goat anti-rabbit or anti-mouse IgG (1:10,000) secondary antibody, rocking for 1 hour at room temperature, and washed three times for 5 minutes in TBST. The membrane was then scanned using a Li-Cor Odyssey 9120 Imaging System.

eNOS-lysate reaction conditions
Reactions to determine eNOS activity in eNOS expressing HEK293 cellular lysate were carried out. Lysate extracted as above was supplemented with 2 mM L-arginine, 1 mM NADPH, 20 µM BH4 in solution containing 3 mM dithiothreitol, and 500 nM WT-or nitroTyr-CaM. For those reactions containing Ca 2+ or EGTA, they were supplemented with 10 mM of either, while those reactions supplemented with L-NAME to a concentration of 1 mM. The reaction was initiated by the addition of the NOS cofactors listed above and incubated for 30 minutes at 37 °C. The eNOS reactions were carried out at a 50 µL scale in triplicate and stopped via freezing in dry ice.

eNOS-activity assays
The amount of NO produced in lysate was measured amperometrically by an AmiNO700 NO selective electrode (Innovative Instruments, Inc.) as described previously (35).         The two sites of physiological tyrosine nitration in calmodulin are shown, Tyr99 in yellow and Tyr138 in orange, and Ca 2+ (shown as green spheres corresponding to the van der Waals radius). Hydrogen bonding between Glu82 in purple and Tyr138 is key to structural coupling between the N-and C-terminal lobes of CaM. Nitration of Tyr138 lowers the pKa of the residue resulting in a weakened hydrogen bond, likely lowering interlobe coupling. The carbonyl oxygen of Tyr99 (yellow) involved in chelating Ca 2+ in EF-hand III is also shown. A gainof-function may occur if holoCaM (PDB 3CLN) binds to a target protein, like eNOS, with greater affinity (eNOS-CaM complex, PDB 1NIW). Further, a gain-of-function could occur if nitroTyr-CaM binding induces increased target protein activity. Nitration of these sites leads to different effects on eNOS, the gain-of function change caused by nitroTyr-CaM-99 leads to decreased activity of eNOS, while the gainof-function caused by nitroTyr-CaM-138 leads to increased activity of eNOS.

CaM tyrosine nitration effects on eNOS
Values represent the average of 3 measurements ± SD, *from t test as compared to WT