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Published in final edited form as: FEBS Lett. 2012 Dec 22;587(3):297–301. doi: 10.1016/j.febslet.2012.12.012

Calmodulin-induced structural changes in endothelial nitric oxide synthase

Anthony Persechini 1,*, Quang-Kim Tran 1, DJ Black 1, Edward P Gogol 1
PMCID: PMC3569036  NIHMSID: NIHMS431209  PMID: 23266515

Abstract

We have derived structures of intact calmodulin(CaM)-free and CaM-bound endothelial nitric oxide synthase (eNOS) by reconstruction from cryo-electron micrographs. The CaM-free reconstruction is well fitted by the oxygenase domain dimer, but the reductase domains are not visible, suggesting they are mobile and thus delocalized. Additional protein is visible in the CaM-bound reconstruction, concentrated in volumes near two basic patches on each oxygenase domain. One of these corresponds with a presumptive docking site for the reductase domain FMN-binding module. The other is proposed to correspond with a docking site for CaM. A model is suggested in which CaM binding and docking position the reductase domains near the oxygenase domains and promote docking of the FMN-binding modules required for electron transfer.

Keywords: nitric oxide synthase, calmodulin, enzyme regulation, enzyme structure

INTRODUCTION

The nitric oxide synthases (NOS) catalyze formation of NO and L-citrulline from L-arginine and oxygen, with NADPH as the electron donor [1]. The three major mammalian forms of the enzyme are commonly referred to as iNOS (inducible), nNOS (neuronal) and eNOS (endothelial) [2,3]. All of these are functional homodimers of 130 – 160 kDa monomers. Each monomer contains a reductase and oxygenase domain joined by a ~35 amino acid linker sequence containing a calmodulin (CaM)-binding domain [1]. The interface between the two oxygenase domains appears to be responsible for maintenance of the enzyme dimer in solution [4–7]. Each reductase domain contains an FMN-binding and FAD-NADPH-binding module, joined by a ~20 amino acid linker [8]. Electron transfer during catalysis occurs in trans, with electrons flowing from the reductase domain of one monomer to the heme reaction center in the oxygenase domain of the other [9–11]. This appears to involve movement of the FMN modules between their respective NADPH-FAD modules and docking sites on the oxygenase domains [12,13].

All three enzyme isoforms have negligible synthase activity in the absence of CaM, which is bound with significant affinity to eNOS and nNOS only in its Ca2+-bound form, and to iNOS in both its Ca2+-free and Ca2+-bound forms [14–17]. Various observations suggest that CaM activates synthase activity both by increasing the efficiency of electron transfer within the reductase domains, from NADPH to FMN via FAD, and by increasing the efficiency of electron transfer to the heme reaction centers via reduced FMN, in part by mobilizing the FMN modules [12,13,18–21]. Although crystal structures have been determined for the dimeric eNOS oxygenase domain [7], and for a dimeric form of the nNOS reductase domain [8], no structures have been published thus far for an intact NOS isoform.

In this paper we present solution structures of full-length CaM-bound and CaM-free bovine eNOS at a nominal resolution of ~25 Å, derived by reconstruction from cryo-electron micrographs of the enzyme in vitreous ice. These structures suggest significant new insights to the structural relationship between the reductase and oxygenase domains, and how this relationship is affected by CaM to produce synthase activation.

MATERIALS AND METHODS

A mutant of bovine eNOS containing a phosphomimetic S1179D substitution was used for these investigations because its maximal CaM-dependent synthase activity is twice that of the native protein, suggesting that in the presence of CaM more is in the fully active conformation [22]. This protein was expressed in E. coli and purified as described elsewhere [22,23]. The vertebrate CaM amino acid sequence, encoded by a rat cDNA, was expressed in E. coli and purified as described previously [24]. Immediately prior to preparation of samples for microscopy, 50 μL aliquots of purified eNOS were thawed and analyzed by size exclusion chromatography on a Superdex 200 HR 10/30 column at 4 °C in a buffer containing 25 mM Tris-HCl, pH 7.4, 100 mM KCl, 1 mM CaCl2 and 1 mM dithiothreitol. Peak fractions previously shown to correspond with the intact dimeric enzyme were pooled, and the monomer concentration of eNOS was determined based on optical absorbance at 397 nm [23]. Prior to freezing on grids, the enzyme was diluted to a concentration of 30 to 150 nM in column buffer, with or without a 1.5-fold molar excess of (Ca2+)4-CaM. The apparent KD for the (Ca2+)4-CaM-eNOS complex is below 1 nM [25], so under these conditions the enzyme should be saturated with CaM.

Fenestrated carbon films (Quantifoil Micro Tools GmbH) subjected to glow-discharge were used for application, blotting and freezing of proteins in liquid ethane. The samples were stored in liquid nitrogen until loading into a Gatan 626 holder and imaging with a JEOL 1200 IIX electron microscope at 100 KV, using minimal dose protocols. Micrographs were recorded on Kodak SO163 film, using defocus values between 1.2 and 3 μm, and digitized using a Hi-Scan drum scanner with a 5 Å pixel on the specimen. Individual particles were selected from images wavelet-filtered to increase contrast, and the coordinates thus obtained were used to extract unfiltered particle images in 40 × 40 pixel (200 × 200 Å) boxes. The CaM-free and CaM-bound eNOS data sets each contain ~25,000 images. Phase correction of the particle images was based on defocus values estimated using the ACE software package [26].

Euler angles were assigned to each image based on projection-matching to de novo common-lines initial models. These were generated from reference-free image averages of both data sets sorted into classes by iterative multivariate statistical analysis with the EMAN software package [27]. An initial model derived in this manner for each data set (+/− CaM) was used to initiate iterative projection- matching in 7° angular increments with two-fold symmetry imposed, using the EMAN software. A cutoff for correlation with model projections eliminated approximately 35% of the particles from the data. Convergence was reached within five to eight rounds of refinement based on round-to-round resolution calculations. To test model dependence, the two initial models (+/− CaM) were exchanged, and the final reconstructions of each dataset were visually indistinguishable from those initiated with the “correct” model. The amplitudes of the reconstructions were corrected in defocus groups guided by a solution scattering curve of a similar-sized protein dimer, fatty acid synthase [28], at resolutions between 100 and 25 Å. The resolutions of the reconstructions were calculated by comparison of Fourier shell coefficients (Fig 1A), both yielding a limit of~25 Å at a 0.5 correlation value. Characteristic projections of both reconstructions compare well with reference-free class averages of the phase-corrected data derived using the refine2d component of the EMAN software package (Figs 1B and C).

FIG 1. Analysis of reconstructions.

FIG 1

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(A) Fourier shell correlation (FSC) plots for the CaM-free (○) and CaM-bound (●) reconstructions derived from odd and even images in the projection classes. Nominal resolutions correspond to the reciprocal of the shell where the FSC value equals 0.5, which yields a limiting value of ~25 Å for both reconstructions. (B & C) Characteristic projections (P) of the CaM-free (B) and CaM-bound (C) eNOS reconstructions compared with reference-free class averages (RF) derived directly from the data. Projections of the reconstructions before amplitude correction were generated after aligning them (corr = 0.96) using the Chimera software [29]. Reference-free class averages were derived from phase-corrected images as described in Materials and Methods.

Fitting and correlation of a simulated 25 Å resolution density map derived from the oxygenase domain dimer crystal structure (PDB ID = 1FOP) [7] was performed using the Chimera molecular graphics package [29]. The DelPhi software suite [30] was used to calculate the electrostatic potential surface for the crystal structure displayed in Fig 1C. A homology model for the eNOS FMN module was derived using standard methods from the nNOS reductase domain dimer crystal structure (PDB ID = 1TLL) [8].

RESULTS

The final CaM-free and CaM-bound eNOS reconstructions are displayed in Figs 2A and B as volumes enclosed at the level of steepest density drop-off, which corresponds with the apparent surface of the protein. A simulated 25 Å density map (colored red) for the oxygenase domain dimer has been fitted to the CaM-free and CaM-bound reconstructions [7]. The reconstructions and simulated oxygenase density are also represented in the figure as cross-sectional contour plots taken at the levels indicated in the reconstructions by lines 1 – 4. The red lines in the contour plots enclose the simulated density for the fitted oxygenase domain dimer.

FIG 2. 3-D representations and cross-sectional contour plots of eNOS reconstructions.

FIG 2

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(A and B) Surface representations of CaM-free (A) and CaM-bound eNOS reconstructions are shown in gray in the first row. A simulated density map for the oxygenase domain dimer fitted into the reconstructions is represented red. Cross-sectional contour plots representing evenly spaced densities are also presented, taken at the levels indicated in by the numbered lines. The fitted simulated oxygenase density is outlined in red. (C) The CaM-bound reconstruction displayed with an electrostatic potential surface derived from the fitted oxygenase domain dimer illustrating the basic patches (colored blue) overlapped by volumes f and c.

Both the CaM-free and CaM-bound reconstructions accommodate the oxygenase dimer crystal structure, but contain little additional volume to fit the reductase domains, which correspond to ~60% of the 260 kDa mass of the eNOS dimer. An excellent correspondence can be obtained between the CaM-free reconstruction and the simulated oxygenase domain density (correlation = 0.93). However, the reductase domains appear to have been lost by the image averaging inherent in the reconstruction process, indicating that they are mobile with respect to the oxygenase domains. The small amount of volume near the the “top” of the CaM-free reconstruction that is unaccounted for by the simulated oxygenase density may correspond with ~65 amino acids missing from the N-termini of the crystal structure [7].

In the case of the CaM-bound eNOS, the highest-density portions of the reconstruction appear to match the simulated oxygenase density, but extensive peripheral density cannot be accounted for by CaM alone, which at 16.8 kDa has only ~10% of the mass of a synthase monomer. This additional density is therefore likely to correspond with the reductase domains, suggesting they are less mobile in the presence of CaM. It appears to be concentrated in two volumes on the surface of each oxygenase monomer, labeled f and c in Fig 2B. As seen in Fig 2C, volume f overlaps a basic patch on the oxygenase domain that has been proposed to participate in docking of the FMN module during catalysis [12,13,21,31,32]. Volume c overlaps a second basic patch, which we propose corresponds with a docking site for bound CaM. In spite of these density concentrations, the diffuse nature of much of the additional density visible in the CaM-bound reconstruction suggests that significant portions of the reductase domains remain mobile.

DISCUSSION

The low-resolution structures of intact eNOS presented here demonstrate that the oxygenase domain portion of the eNOS dimer can be readily identified in solution by its correspondence to the X-ray crystal structure of the oxygenase domain dimer. However, the reductase domains, which comprise approximately 60% of the eNOS molecule, appear to be mobile with respect to the oxygenase domains, especially in the absence of CaM (Fig 2A). In the absence of CaM mobility is sufficient to result in diffusion of almost all the reductase domain density by the reconstruction process. The apparent mobility of the reductase domains is presumably permitted by the ~35 amino acid linker between reductase and oxygenase domains, which could span an average distance of as much as ~50 Å [33]. A crystal structure for a dimeric form of the nNOS reductase domain suggested a model for the intact enzyme in which oxgenase and reductase domain dimers associate in a stacked arrangement [8]. Our results are not consistent with such a model for eNOS. They instead suggest that while the oxygenase domain dimer is maintained in solution, the reductase domains are highly mobile with respect to it.

Additional protein is visible in the CaM-bound reconstruction that most likely corresponds to portions of the reductase domains. At the resolution of this analysis, we cannot definitively identify the elements that comprise these structural additions. However, volume f is posited as the FMN module, due to its overlap with a basic patch on the oxygenase domain previously proposed to participate in CaM-dependent docking of this module (Fig 2C) [21]. Volume c overlaps an additional basic patch on the oxygenase domain (Fig 2C). A crystal structure determined for the complex between CaM and an iNOS fragment containing the FMN module and CaM-binding domain indicates that bound CaM and FMN module are separated by a short ~7 amino acid linker, a feature that appears to be conserved in the synthases [31]. Thus, based on its proximity to the presumptive FMN module, as well as its size, volume c is consistent with bound and docked CaM. Docking of CaM in this manner would be expected to promote what otherwise appears to be an intrinsically weak interaction between the FMN module and the adjacent oxygenase domain [12,13,34].

Molecular modeling was performed to investigate the structural feasibility of our interpretations of volumes f and c in the CaM-bound reconstruction (Fig 3). For the sake of clarity only one of the two symmetry-related pairs of f and c volumes is considered. Three orthogonal views of the model are presented alone (D–F) and superimposed on a surface representation of the CaM-bound eNOS reconstruction (A–C). The FMN module is positioned to achieve a reasonable correspondence with volume f on the surface of the green oxygenase monomer. It is depicted in magenta to indicate that it is continuous with the magenta oxygenase domain, and is therefore positioned in the model for the requisite transfer of electrons in trans to the heme in the adjacent oxygenase domain (colored green). The 18.9 Å distance between FMN and the reaction center heme iron in this domain achieved in the model is similar to the 18.8 Å distance estimated based on pulsed EPR data [32]. The complex between (Ca2+)4-CaM and the eNOS CaM-binding domain is positioned adjacent to the magenta oxygenase domain in volume c, with the CaM-binding domain colored magenta to indicate its continuity with this domain. Bound CaM is colored cyan and bound Ca2+ ions are indicated by green spheres. The short linker between the FMN module and the C-terminus of the CaM-binding domain is modeled as a random coil, demonstrating that it can span the distance between volumes f and c, as required for the FMN module to dock with the oxygenase domain in this model (Fig 3).

FIG 3. Molecular modeling of oxygenase and projections in CaM-bound eNOS.

FIG 3

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Orthogonal views of the fitted oxygenase domain dimer and modeled FMN module and CaM complexes are shown with (A – C) and without (D – F) display of the CaM-bound reconstruction. The oxygenase is depicted as a α-carbon tracing, with the two monomers (residues 67–482 and 69–482) colored green and magenta. The reaction center hemes are displayed as hard sphere models. The FMN module (residues 520–716) is depicted in magenta, to indicate that it belongs to the same monomer as the magenta oxygenase domain. Bound FMN is displayed as a hard sphere model. The CaM-binding domain (residues 494–512) is colored magenta, as it also belongs to the same monomer. Bound CaM is colored cyan, and bound Ca2+ ions are indicated by green spheres. The linker (GTLMAKR) between the FMN module and the CaM-binding domain is modeled as random coil. The linker between the magenta CaM-binding and oxygenase domains (KGSATKGAGIT) is not shown.

Our results are consistent with a minimal two-step model for CaM-dependent synthase activation (Fig 4). In the first step, CaM binds a CaM-binding domain in the enzyme, activating the reductase domain and mobilizing the FMN module. In the second step, CaM docks on the oxygenase domain that is continuous with the CaM-binding domain. This promotes docking of the FMN module with the adjacent oxygenase domain. The FAD-NADPH module is depicted as remaining mobile with respect to the oxygenase domain dimer, and this may account for some of the diffuse density observed in the presence of CaM. The idea that CaM activates synthase activity through minimally a two-step process is not new, although our results support a novel CaM-docking hypothesis for the second step [12,18,34]. We have previously demonstrated that a similar binding and docking mechanism applies to CaM-dependent activation of skeletal muscle myosin light chain kinase, with the docking step apparently required to allow substrate access to the enzyme active site [35,36].

FIG 4. A two-step model for CaM-dependent activation of the constitutive nitric oxide synthases.

FIG 4

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Aside from the two oxygenase domains (colored magenta and green), only one of two symmetry related sets of elements is depicted for the sake of clarity. Reaction center hemes are represented by diamonds. Auto-inhibitory sequences in the reductase domain that may interact with the CaM-binding domain in the absence of CaM are not represented [12,18]. In step I, binding of Ca2+-CaM (c) activates the reductase domain and releases the FMN module (f) from the NADPH-FAD module (n). In step II, docking of bound CaM with the magenta oxygenase domain positions the reductase domain as required to promote docking of the FMN module with the oxygenase domain in the other synthase monomer (green).

Highlights.

  • Structures of intact eNOS +/− CaM have been derived from cryo-electron micrographs.

  • The reductase domains appear to be mobile with respect to the oxygenase domain dimer.

  • Densities consistent with oxygenase-docked FMN modules are observed in the presence of CaM.

  • Additional densities are observed that are proposed to be bound and docked CaM.

  • A novel model is suggested in which docking of CaM promotes docking of the FMN modules.

Acknowledgments

The authors would like to thank Benjamin Iwai and Leanne Szerszen for expert technical assistance. This work was supported in part by NIH grant GM074887 to A. P. and by a University of Missouri Research Board grant to E. P. G.

Abbreviations

NOS

nitric oxide synthase

eNOS

endothelial NOS

iNOS, nNOS

inducible and neuronal NOS

CaM

calmodulin

Footnotes

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