Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 4754
doi:10.1111/j.1745-7270.2008.00369.x
Preliminary computational modeling of
nitric oxide synthase 3 interactions with caveolin-1: influence of exon 7
Glu298Asp polymorphism
Mandar S. Joshi and John
Anthony Bauer*
Center for
Cardiovascular Medicine, The Research Institute at Nationwide Children’s
Hospital, Columbus Ohio 43205, USA
Received: July 18,
2007
Accepted: September
13, 2007
*Corresponding
author: Tel, 1-614-722-4984; Fax, 1-614-355-3444; E-mail, [email protected]
The significance
of endothelial nitric oxide synthase 3 (NOS3) activity has been recognized for
many years, however it was only recently that the complicated regulation of
this constitutively expressed enzyme in endothelial cells was identified. A
critical component of the NOS3 regulatory cycle in endothelial cells is its
intracellular localization to caveolae. The caveolar coordination of NOS3, more
specifically its interaction with caveolin-1 (Cav-1), plays a major role in
normal endothelial NOS3 activity and vascular bioavailability of nitric oxide.
We have recently shown that the presence of NOS3 exon 7 Glu298Asp polymorphism
caused diminished shear-dependent NOS activation, was less extensively
associated with caveolae, and had a decreased degree of interaction with
Cav-1. Here, we carried out preliminary investigations to identify possible
mechanisms of the genotype-dependent endothelial cell responses we observed in
our previous investigations. Through this approach we tested the hypothesis
that computer simulations could provide insights regarding the contribution of
this single nucleotide polymorphism to regulation of the NOS3 isoform. We
observed that in the Glu/Asp and Asp/Asp mutant genotypes, the amount of NOS3
associated with Cav-1 was significantly lower. Additionally, we have shown,
using a theoretical computational model, that mutation of an amino acid at
position 298 might affect the protein-protein interactions and localization
of the NOS3 protein. These alterations might also affect the protein function
and explain the enhanced disease risk associated with the presence of
Glu298Asp polymorphism in the NOS3 protein.
Keywords NOS3; caveolin-1; gene polymorphism; computational modeling;
protein-protein interaction
The vascular endothelium is highly metabolically active and plays a
key role in vascular homeostasis through the release of a variety of
substances, including the vaso-regulator nitric oxide (NO) [1]. The actions of
NO can be altered severely under various conditions of stress, such as hypoxia,
oxidants, reactive nitrogen species, and shear stress [2,3]. Diminished NO
availability has been associated with several settings of endothelial
impairment, including acute hypoxic or hyperoxic organ injury and chronic forms
of vascular disease [3–8]. NO synthase 3 (NOS3) is important for maintenance of systemic
blood pressure, vascular remodeling, angiogenesis, and wound healing [9–12]. A critical component of the NOS3 regulatory cycle in endothelial
cells is its intracellular localization to caveolae [13–15]. Caveolae are 50–100 nm
flask-shaped, cholesterol- and glycosphingolipid-rich membrane microdomains in
the plasma membrane that provide intracellular coordination of many
membrane-associated proteins and putatively function as regulators of
cholesterol transport, endocytosis, and signal transduction [16,17]. In
endothelial cells, caveolin-1 (Cav-1), a 22-kDa protein that coats the
cytoplasmic surface of this specialized microdomain, is the major protein constituent
of caveolae and is thought to hold or “store” NOS3 in an inactive
state, wherein phosphorylation causes dissociation and NOS3 catalytic
activation [18]. The caveolar coordination of NOS3, more specifically its
interaction with Cav-1, plays a major role in normal endothelial NOS3 activity
and vascular bioavailability of NO and is a key controller of shear-dependent
NO release [19]. Several specific allelic variations of the NOS3 gene are known
to date, with many of these having been linked to increased cardiovascular
disease risk [20–27]. Out of a total of eight variations in the human NOS3 gene,
only one codes for a variant form of the enzyme; a single nucleotide
polymorphism (SNP) (G>T) at position 894, leading to the sequence change
Glu298Asp. Using a “candidate” gene approach, in which patient
genotypes are related to the incidence of a disease and/or clinical outcome to
test for statistically significant associations (parametric and/or
non-parametric linkage analyses), many reports have indicated that the NOS3
Glu298Asp polymorphism is associated with increased occurrence of
cardiovascular disease, including coronary artery disease [25,28,29],
myocardial infarction [30,31], hypertension [32,33], and stroke [34]. Although
the exact biological consequences of this specific variation are not
identified, prior studies by others using isolated NOS3 enzyme derived from
transfected COS7 cells indicated no difference in purified enzyme catalytic
activity between wild-type (Glu/Glu) and variant (Asp/Asp) NOS3 [35]. Given that caveolar associations have been recognized as important
for normal NOS3 performance in endothelial cells, and the established importance
of this variation for disease risk in humans, we recently investigated the
impact of the NOS3 Glu298Asp polymorphism on endothelial cell responses to
shear stress and caveolar localization. We found initial evidence that the
altered enzyme, when in its native endothelial cell environment, has a
diminished shear-dependent NOS activation, is less extensively associated with
caveolae, and has a decreased degree of interaction with Cav-1 [36]. Our
preliminary findings using protein biochemistry techniques thus suggested that
the Glu298Asp variation might influence protein-protein interactions. In this
project we carried out preliminary computer modeling to further investigate
possible mechanisms of the genotype-dependent endothelial cell responses we
observed in our previous investigations [36]. Through this approach we tested
the hypothesis that computer simulations could provide insights regarding the
contribution of this SNP to regulation of the NOS3 isoform.
Materials and Methods
NOS3 genotyping
Human umbilical vein endothelial cells were screened for NOS3
genotype using polymerase chain reaction-based DNA amplification followed by
restriction enzyme digestion as described previously [30,36].
Cell lysis and
immunoprecipitation
After washing three times with ice-cold phosphate-buffered saline,
cells were lysed in NP-40 lysis buffer containing 1% NP-40, 50 mM Tris, pH 7.5,
150 mM NaCl, 5 mM EDTA, 0.5 mM Na3VO4, 50 mM
NaF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 10 mg/ml pepstatin A, and 1 mM phenylmethylsulphonyl fluoride. The cell
extracts were used for immunoprecipitation reactions. The protein G immunoprecipitation
kit from Sigma (St. Louis, USA) was used. The cell extracts were incubated
overnight at 4 ?C, with NOS3 polyclonal antibody (pAb) on protein G agarose
beads. The beads were washed thoroughly after incubation and the sample was
prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The
blots were probed for Cav-1 protein. The amount of Cav-1 associated with NOS3
was calculated as a ratio of Cav-1 band optical density to NOS3 band optical
density. The optical density analysis was carried out using LabWorks image
analysis software (version 4.6; UVP-Media Cybernetics, Upland, USA).
Western blotting
Samples prepared as described above were separated on 4%–20% Tris-glycine
gel. Standard Western blotting protocols were used as described previously
[36].
Protein structures and
computational modeling
The amino acid sequence of human NOS3 (accession No. AAH69465) was
retrieved from the National Center for Biotechnology Information protein
sequence database (http://www.ncbi.nlm.nih.gov/). Computational modeling
was carried out using the DeepView software Swiss-PdbViewer [37]. The NOS3
protein amino acid sequence was loaded into DeepView software and the 3-D
structure was generated. Using the mutation feature in the software, variation
of the amino acid at position 298 (Asp) was done in chain B of the protein.
Chain A of the protein had the wild-type amino acid Glu at position 298.
Visualization of the protein structure was carried out in the secondary
structure form. Specific amino acid residues required for enzyme function were
identified in the protein 3-D structure and amino acid distances were calculated
using the distance measurement feature of the PdbViewer software. Using the
amino-acid visualization tool in the software, specific amino acids of interest
were selected one at a time and distance measurements were carried out from
amino acid 298. All the measurements from amino acid 298 were done from the
side chain carbon atom. All distances were measured in angstrom units () and
are expressed here as absolute values and percentage variation.
Confocal microscopy
Cells were grown on a collagen-coated slide to confluence. They were
fixed with formalin and washed with Tris-buffered saline. Double labeling was
done using rabbit anti-NOS3 pAb (Transduction Biolabs, Bedford, USA) and rabbit
anti-Cav-1 pAb (Upstate Biotechnology, Lake Placid, USA). Chicken anti-rabbit
(Alexa Fluor 488; Molecular Probes, Carlsbad, USA) labeled antibody was used as
a secondary for NOS3 primary antibody, which showed green fluorescence. Goat
anti-rabbit F(ab)2 fragment (Alexa Fluor 633; Molecular Probes) labeled
antibody was used as a secondary for Cav-1 primary antibody, which showed red
fluorescence. Confocal microscopic observations were done using the LSM Meta
510 laser confocal microscope.
Statistical analysis
All data are expressed as mean±SEM. Each cell line was treated as an
individual sample. GraphPad Prism software (version 4.0; San Diego, USA) was
used to fit the data and carry out the statistical analysis. A simultaneous
comparison of groups was handled by one-way anova, with Student-Newman-Keuls post-hoc
analysis. For analyzing the combination effects, two-way anova
was used to test differences between the genotypes and various
treatments. In all cases, significance was defined as P<0.05.
Results
Identification of key amino
acid residues
Using the National Center for Biotechnology Information sequence
database and previous reports in the literature, key amino acids essential for
NOS3 enzymatic activity were identified. These amino acid residues are listed
in Table 1, and were used for theoretical distance calculations in the
computational modeling studies.
Basal Cav-1/NOS3 association
is lower in the variant genotypes
Initially, immunoprecipitation studies were carried out and
quantified as described in “Materials and Methods”. The ratio of
Cav-1/NOS3 band optical density was 2.53±0.15 in Glu/Glu, but it was
significantly lower in Glu/Asp (1.57±0.31) and Asp/Asp (1.36±0.23) variant
genotypes (P<0.05) (Fig. 1). These results show that
under basal conditions the NOS3/Cav-1 association is altered in the Asp variant
genotypes.
Basal NOS3 membrane
localization diminished in the Asp variant genotypes
We also carried out confocal microscopy under basal conditions and
probed for co-localization of NOS3 and Cav-1. We observed greater co-localization
of NOS3 and Cav-1 at the cell membrane in the wild-type cells. These studies
showed that under basal conditions, the Glu/Glu wild-type cells have a greater
NOS3 localization at the caveolar regions on the cell membrane as compared to
the Glu/Asp and Asp/Asp variant endothelial cells (Fig. 2).
Altered protein structural
geometry in the variant amino acid chain
The 3-D protein structure was constructed using DeepView software
[37]. The important substrate and co-factor binding sites were highlighted and
shown in Fig. 3. Using the software, the amino acid at position 298 in
chain B was mutated from Glu to Asp. Individual amino acid distances from
various substrate and co-factor binding sites were calculated for chain A (Glu
at position 298) and chain B (Glu at position 298) based on this 3-D structure
(Fig. 4). The absolute distances are shown in Table 2 and
represented as angstrom units () as well as the percentage difference.
Variation of the amino acid residue at position 298 from Glu to Asp in chain B
resulted in altered protein geometry. The distance between the important sites
and altered residues were consistently lower in chain B compared to chain A.
The percentage differences were greatest in the Cav-1 binding region when amino
acid variation occurred at position 298.
Discussion
Theoretical protein modeling serves as an important tool for
structure elucidation and prediction of protein interactions and structural features
[38]. Since the identification of the NOS family of enzymes, many structural
features have been elucidated. Based on previous reports we compiled a list of
the important sites that are necessary for NOS3 protein function [39–41]. NOS3 is
regulated in a complex manner and requires many post-translational
modifications for proper functioning. Myristoylation and palmitoylation at the
N-terminal are important for proper localization and targeting of the protein
to organelles in the cell [42]. NOS3 enzyme function is also dependent on
substrate (L-arginine) and co-factor (tetrahydrobiopterine, Zn)
availability as well as their interactions with other known regulatory
proteins (Cav-1 and Hsp90) [13]. Given the importance of tight
regulation of NOS3 protein and its important interactions with Cav-1 protein,
we carried out NOS3 immunoprecipitation in endothelial cells that were
previously genotyped for the presence of Glu298Asp polymorphism. We observed
that in the Glu/Asp and Asp/Asp variant genotypes, the amount of NOS3
associated with Cav-1 was significantly lower. These observations were
recapitulated by the confocal microscopic observations. Moreover, using a
caveolar membrane-enrichment method we also showed that the amount of NOS3
present in the caveolar membrane is significantly lower in the variant
genotypes. These data indicate that NOS3 is not localized properly to the
caveolar membrane and its interaction with Cav-1 protein is altered in the
Glu/Asp and Asp/Asp variants.
NOS3 is regulated in a cyclic manner and under static conditions,
and it is bound to membrane caveolae through its interactions with Cav-1. On
stimulation, this interaction is dissociated and NOS3 enters the cytosol where
it interacts with other proteins and co-factors to produce NO. Thus,
Cav-1-bound NOS3 represents the available pool of NOS3 that can readily produce
NO in the cell.The 3-D protein structures might provide valuable insights into the molecular basis of protein function. Combining sequence information with the 3-D structure might give detailed information
regarding the structural relevance of variations and protein function [43].
Traditional methods like X-ray crystallography and nuclear magnetic resonance
spectroscopy are time-consuming and require a pure form of the protein. We used
the Swiss-Prot DeepView software to carry out theoretical structural modeling
of the NOS3 protein [37,43,44]. After resolving the structure and marking key
sites on the protein, we observed that the amino acid at position 298 is not
close to the active site pocket of NOS3. The important site for NOS3 function
is the dimerization site that has all the substrate and co-factor binding
regions. Thus it is unlikely that the presence of the mutated amino acid (Asp)
at position 298 will alter the enzymatic function of NOS3. We carried out
computational modeling studies of the protein. Theoretical distance
calculations revealed that variation of the amino acid at position 298 might
affect protein geometry. The variation of an amino acid in one chain or both
chains might introduce a structural variation in the protein structure and
also affect the interactions of the protein. It is unlikely that this change in
protein amino acid distances will affect the enzymatic pocket of the protein,
but it might alter the protein localization and trafficking. It is not
completely understood whether an amino acid distance change of 3–10 ? will
actually make a difference in protein structure, but some reports suggest that
a change of distance by 1 ? might affect the electrostatic energies to a great
extent [45]. We do not know the electrostatic energies in this modeling study,
but it would be interesting to study these in light of the variation at
position 298. Although this is not the most suitable method to characterize
protein structural alterations, it does provide a good “starting
point”. The differences observed by using this method provide some
insights into the structural and functional implications of a gene SNP. These
observations provide preliminary evidence that the changes in protein
structure might affect its interactions with substrate or co-factor. In conclusion, we have shown, using a
theoretical computational model, that variation of an amino acid at position 298
might affect the protein-protein interactions and localization of the NOS3
protein. These alterations might also affect the protein function and might
explain the enhanced disease risk associated with the presence of Glu298Asp
polymorphism in the NOS3 protein.
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