Deciphering the binding of caveolin-1 to client protein endothelial nitric oxide synthase

22 Pages·2014·8.45 MB·English

JBC Papers in Press. Published on March 19, 2014 as Manuscript M113.528695
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M113.528695
Deciphering the binding of caveolin-1 to client protein endothelial nitric oxide synthase (eNOS):
scaffolding sub-domain identification, interaction modeling, and biological significance

Andy E Trane1, Dmitri Pavlov1, Arpeeta Sharma1, Uzma Saqib4, Kelvin Lau2, Filip van
Petegem2, Richard D. Minshall4,5, Linda J. Roman3, Pascal N Bernatchez1

1 Institute for Heart and Lung Health at St. Paul’s Hospital, The James Hogg Research Centre,
and Department of Anaesthesiology, Pharmacology and Therapeutics, or 2 Department of
Biochemistry, University of British Columbia, Canada, 3 University of Texas Health Science
Center at San Antonio, Department of Biochemistry, University of Texas, USA, Departments of
4 Pharmacology and 5 Anaesthesiology , University of Illinois, Chicago, IL
*Running title: eNOS binding to Cav-1 and modulation of NO release
DDDDD
owowowowow
nnnnn
To whom correspondence should be addressed: Pascal N. Bernatchez, St. Paul’s Hospital’s lololololo
aaaaa
ddddd
Centre of Heart and Lung Innovation, Department of Anesthesiology, Pharmacology and ededededed
Therapeutics, University of British Columbia, 1081 Burrard st, room 166, Vancouver (BC) fro fro fro fro fro
mmmmm
Canada, V6Z 1Y6, Phone: [phone] x66060, E-mail: [email protected] http http http http http
://w://w://w://w://w
wwwww
Keywords: Caveolae, Caveolin, Endothelium, Nitric Oxide, Nitric Oxide Synthase wwwww
.jbc.jbc.jbc.jbc.jbc
.o.o.o.o.o
brg/ brg/ brg/ brg/ brg/
yyyyy
Background: One of Cav-1s most interaction, which occurs specifically in g g g g g
uuuuu
significant client proteins is the endothelial vascular endothelial cells, is responsible eseseseses
t ot ot ot ot o
nitric oxide synthase (eNOS). But their for the multiple phenotypes observed in n An An An An A
ppppp
specific binding site is unknown. Cav-1-null animals. Under basal ril 5ril 5ril 5ril 5ril 5
Results: We describe how Cav-1 binds to conditions, Cav-1 binds eNOS and , 2, 2, 2, 2, 2
00000
eNOS and how biologically active NO can inhibits nitric oxide (NO) production via 1919191919
be increased. the Cav-1 scaffolding domain (CAV; aa
Conclusion: We provide the most detailed 82-101). Although we have recently
characterization of eNOS binding to Cav-1. shown that CAV residue F92 is
Significance: Our data provide a deeper responsible for eNOS inhibition, the
understanding of Cav-1 signaling and NO ‘inactive’ F92A Cav-1 mutant
generation in physiological processes. unexpectedly retains its eNOS binding
ability and can increase NO release,
indicating the presence of a distinct
Abstract
eNOS-binding domain within CAV.
Caveolin-1 (Cav-1) gene inactivation
Herein, we identified and characterized a
interferes with caveolae formation and
small 10 aa CAV sub-sequence (90-99)
causes a range of cardiovascular and
that accounted for the majority of eNOS
pulmonary complications in vivo. Recent
association with Cav-1 (Kd = 49nM), and
evidence suggest that blunted Cav-1 /
computer modeling of CAV 90-99
endothelial nitric oxide synthase (eNOS)
docking to eNOS provides a rationale for

  1
the mechanism of eNOS inhibition by interest, endothelial nitric oxide synthase
F92. Finally, using gene silencing and (eNOS) 15. eNOS is responsible for the
reconstituted cell systems, we show that conversion of L-arginine into nitric oxide
intracellular delivery of a F92A CAV 90- (NO), the most critical direct regulator of
99 peptide can promote NO vascular tone due to its capacity to promote
bioavailability in eNOS- and Cav-1- extensive vasorelaxation and act
dependent fashions. To our knowledge, downstream of any constrictive agonist16.
these data provide the first detailed eNOS is post-translationally palmitoylated
analysis of Cav-1 binding to one of its and trafficked to caveolae 17 where it can
most significant client proteins, eNOS. interact with other key signaling proteins
including Cav-1 via its scaffolding domain
INTRODUCTION (CAV; a.a. 82-101). Studies performed
Caveolae, latin for small caves, are using yeast two hybrid screens, GST
microscopic invaginations of the plasma pulldown experiments, and co-
membrane known to regulate key aspects of immunoprecipitations combined with
endocytosis, transcytosis, mechano- enzymatic assays have supported the claim
transduction, and clustering of signaling of direct eNOS docking to CAV 18, 19, Do
w
molecules 1-4. The main coat protein of followed by attenuated NO production. nlo
a
caveolae in non-muscle cells, caveolin-1 Following agonist challenge, eNOS is de
d
(Cav-1) 5, is essential for the biogenesis of thought to dissociate from Cav-1 and get fro
caveolae, as homozygous deletion of the phosphorylated 18 by various kinases such as hm
Cav-1 gene causes complete loss of caveolae Akt 20. Subsequent binding of calmodulin ttp://w
in Cav-1-expressing tissues6. Surprisingly, and HSP90 further transition eNOS into a w
w
Cav-1 knockout animals are viable6, 7 more active state 21, leading to increased NO .jb
c
although their cardiovascular and pulmonary release. Finally, as a mechanism of NO- .org
systems show profoundly aberrant dependent feedback inhibition, we recently by/
g
behaviours, including hyperproliferative showed that eNOS binding specifically to ue
s
vascular endothelium, abnormal Src-phosphorylated Cav-1 (pY14) increases t on
permeability, vascular constriction, and eNOS activity returns to basal level22. Ap
vasodilation, flow-dependent remodelling, ril 5
Since decreased eNOS-derived NO , 2
as well as left and right ventricular cardiac 01
release is considered an independent 9
hypertrophy as a consequence of systemic
hypotension and pulmonary hypertension 3, predictor for a range of pathologies such as
6-11. Together, these data provide irrefutable atherosclerosis23, systemic and pulmonary
arterial hypertension24-27, signaling events
evidence of the role of Cav-1 in caveolae
that result in decreased eNOS activity/NO
assembly but also of the complexity of the
bioavailability are routinely linked to
caveolar system in whole body homeostasis.
cardiovascular and pulmonary diseases28-30.
In addition to its caveolae formation Interestingly, recent transgenic experiments
abilities, Cav-1 acts as a scaffold and have shown that endothelial cell-specific
signaling protein known to interact with a reintroduction of Cav-1 in Cav-1-null
wide variety of client proteins. In animals rescues eNOS localization and
cardiovascular cells and tissues, studies have activity, and that endothelial Cav-1 or eNOS
shown direct caveolin binding to and inhibition rescue most of the cardiovascular
modulation of growth factor and G-protein- and pulmonary defects observed in Cav-1-
coupled receptors 5, 12, protein kinase A 13, null animals8, 31-33, suggesting that eNOS
adenylyl cyclase, Rab5 14 and, of particular likely is the most significant target of Cav-1

  2
binding. These contentions are further Recombinant protein expression and
supported by observations that eNOS-null purification. GST proteins were expressed
mice are resistant to the therapeutic effects in BL21 bacterial cultures as previously
of a Cav-1-mimicking cell permeable described37. All concentrations are in
peptide34, 35, further stressing the importance reference to the volume of solution at each
of the Cav-1/eNOS interaction in whole step. Samples were centrifuged at 4ºC at
body homeostasis. 16,000 g for 30 mins. The supernatant was
stored at -80ºC until required. The
In an attempt to deepen our
baculovirus-derived bovine eNOS used for
understanding of the mechanism of eNOS
GST pull-downs was from Cayman
inhibition by Cav-1, we recently performed
Chemicals, whereas highly purified eNOS
experiments using an F92A mutant of Cav-1 protein of bacterial origin 38 was used for
previously shown by our team to have lost
fluorescence experiments and to reproduce
its eNOS inhibitory activity and we
GST pull-down results.
unexpectedly observed that F92A Cav-1 can
increase NO release while and has retained GST-fusion protein resin preparation.
its eNOS-binding property 35, 36. This Glutathione-coated agarose resins (Pierce) D
o
suggested the presence of a distinct eNOS- were briefly washed with STET (STE with w
n
lo
binding sub-domain within CAV. Herein, 1% triton-X) and pelleted via centrifugation. a
d
e
d
we perform detailed characterization of The wash solution was replaced with fro
eNOS / Cav-1 binding - independently of supernatant prepared above. The mixture m
h
Cav-1’s ability to inhibit eNOS activity 36 - was rotated at 4ºC for 1 hr and centrifuged. ttp
and identify the molecular determinants of Resins were washed with STET and ://w
w
w
this interaction. Due to the importance of centrifuged. This cycle was repeated 3 .jb
c
NO production in cardiovascular and times. All centrifugation steps were .o
rg
pulmonary health, our data may lead to performed at 1,300 g (4ºC). Resins were re- b/
y
novel NO-based therapies for eNOS-related suspended in STET and stored at 4ºC. g
u
e
s
disease. t o
Protein pull-down and Immunoblotting. n A
p
The appropriate volume of GST-fusion ril 5
products was dispensed, by using , 2
0
EXPERIMENTAL PROCEDURES SimplyBlue (Invitrogen) protein stain, to 19
Plasmids and constructs. Primers for the gauge relative yields. Discrepancies in bead
respective recombinant GST plasmids were volume were compensated for by blank
purchased from Sigma Aldrich with BAM resins to account for residual trapped
HI and NOT I restriction sites at the 5’ and proteins. Recombinant eNOS was diluted to
3’ ends, respectively. Polymerase chain a concentration of 200 µg/mL in binding
reaction was used to amplify the mutants, buffer (50 mM Tris-HCl, 20% glycerol, pH
using a GST-Cav template encoding for 7.4). GST resins were incubated with the
residues 61-101 of the Cav-1 protein which eNOS solution for 2 hrs at 4ºC and was
were inserted into pGEX-4T-3 GST vectors subsequently washed 5 times using a high
as previously described37. Purified vectors salt wash buffer (150 mM NaCl, 50 mM
were sent to UBC-NAPS for confirmation of Tris-HCl, 1mM EDTA, pH 7.7). The salt
sequence before being transformed into buffer was then replaced with the SDS-
BL21 for protein expression as described. PAGE loading buffer and ran on a split
gradient (7.5 and 12%) acrylamide gel.
Rabbit anti-GST (Invitrogen) and mouse

  3
anti-eNOS (BD transduction) were used to Molecular modeling of peptides. The
detect the respective proteins. Gels were sequence from the wild type and mutant
imaged using our Licor system. peptides were run for NCBI protein blast to
search for proteins having similar sequences.
Polarization assay. eNOS was diluted to
The Crystal structure of NmrA-like family
varying concentrations in buffer containing
domain containing protein 1 (PDB: 2WMD),
50 mM inorganic phosphate, 50 mM KCl,
showing a very high (75%) sequence
and 5 mM DTT (pH 7.5). A
identity was used for modeling the three-
spectrophotometer was used to determine
dimensional structure of peptides. The
the concentration of the fluorescein-labelled
modeled structures of peptides were energy
peptide using the extinction coefficient for
minimized using CharMM forcefiled
fluorescein (ε = 79,000 M-1). Peptides
492 nm (www.charmm.org) in Accelerys Discovery
(100 nM) were added to samples containing
studio (http://accelrys.com/), to relax any of
varying concentrations of eNOS and
the disordered regions in the peptides.
incubated for 1 hr at room temperature.
Samples were then plated on 384-well Molecular docking of peptides into eNOS.
Grenier Bio-One solid black microplates in The crystal structure of heme-containing D
o
triplicates. Polarization was measured with a human endothelial nitric oxide synthase w
n
lo
Tecan Infinite M1000 Pro apparatus in (eNOS) (PDB: 1M9M) was chosen as the a
d
e
d
fluorescent polarization mode using 470 nm receptor for molecular docking studies. The fro
excitation and 515 nm emission filters. The chain A of the full protein was used for the m
h
obtained results were normalized to the docking studies, followed by addition of ttp
plateau value and fitted using GraphPad hydrogens and further energy minimization ://w
w
w
Prism 5 software. in Discovery studio. Molecular docking of .jb
c
the modeled peptides was performed .o
Cell culture, siRNA, NO accumulation individually into the eNOS structure, using brg/
y
and peptide treatment. Bovine aortic the GRAMM-X g
u
endothelial cell (BAECs) were treated with es
server(http://vakser.bioinformatics.ku.edu/re t o
peptides to assay for NO accumulation, in sources/gramm/grammx). Further, top- n A
p
the absence or presence of eNOS siRNA ranking model from each of the docking run ril 5
(Santa Cruz Biotechnology) as previously was chosen as the final docked pose of the , 2
0
described 39, 40. Peptides were purchased peptides. The eNOS-peptide binding was 19
from Elim BioPharm. Adhesion of
further confirmed by KFC2 server
neutrophils was studied by pre-incubating
(http://kfc.mitchell-lab.org/), which predicts
BAEC for 1h with PAF (Avanti Polar Lipids
binding "hot spots" within protein-protein
100 pM), followed by addition of fresh
interfaces by recognizing structural features
DMEM containing Ficoll purified human
indicative of important binding contacts.
neutrophils (5x105 per well of 6 well plate)
Since, peptides are highly flexible as
under static conditions for 15 min, followed
ligands, we further validated the docked
by gentle washing with fresh media and
peptide poses by running an iterative
counting. COS-7-derived NO release was
molecular dynamics. A 10 ns Molecular
studied with a GFP-tagged eNOS plasmid
dynamics (MD) simulation was carried out
and WT Cav-1 adenovirus as described35.
for the top ranking lowest energy structures
NO analysis was performed as previously
given by GRAMM-X server. The resulting
described, whereas nitrate conversion into
conformations were compared with the
nitrite was performed using nitrate reductase
original starting conformations and based on
(Roche).
the superimposition and comparison of the

  4
docked conformations of each, wild-type immunoblot confirmed similar levels of
and mutant peptides before and after the GST resin (data not shown), and was used to
molecular dynamics study, we concluded normalize eNOS levels. This indicated that
that there were little to no deviation among F92 is not a contributor to eNOS binding
them (less then 0.5Å), further validating our and confirms the presence of a yet
docking studies 41, 42 43, 44. unidentified eNOS-binding sub-domain
elsewhere within CAV. However, we
Data and statistical analysis. Immunoblot
observed also that GST-CAV can pull-down
images were quantified with the Odyssey
endogenous Cav-1 (data now shown), a
infrared scanner and software to ensure
likely consequence of the overlapping
quantification linearity and absence of signal
scaffolding and oligomerization domains,
saturation. Values were expressed as a ratio
which suggests that the true eNOS binding
of the density of the eNOS band over the
affinity of GST-CAV 61-101 can be
GST band, and expressed as a percentage of
confounded by the presence of endogenous
the positive control binding. Data for each
Cav-1 containing complexes.
construct were pooled together and were
analyzed using one-tailed t-test to see if The eNOS binding subdomain is located D
o
values differed from 100% (control in the CAV 89-101 section. CAV was w
n
lo
binding). One-way ANOVA analysis with a segmented into 3 sub-domains as previously a
d
e
Dunnett’s post hoc test was used to compare described by our group 45: CAV-A (82-88), d fro
for differences between experimental groups CAV-B (89-95) and CAV-C (96-101; Fig. m
h
for polarization studies while two-way 1B), and deletion or alanine inactivation ttp
ANOVA was used for nitric oxide release. mutants were designed, since poly-alanine ://w
w
w
Significance was defined as P < 0.05 in all sequences tend to form alpha-helixes similar .jb
cases. to the Cav-1 scaffolding domain 46. We c.o
rg
created truncated mutants that encoded for b/
y
RESULTS GST fused to Caveolin-1 a.a 61-81 plus g
u
CAV inhibitory residue F92 does not bind es
CAV-A, GST plus CAV-AB, GST plus t o
eNOS. To confirm the presence and CAV-BC, with an inactivating poly-alanine n A
p
significance of the two sub-domains within sequence replacing CAV-A, and these ril 5
the Cav-1 scaffolding domain (82-101) – an mutant constructs were compared with full- , 2
0
inhibitory sub-domain (F92) and a binding length CAV wild-type (CAV , positive 19
WT
sub-domain (unknown) - we performed a
control) (Fig. 1B) and two negative controls:
pulldown experiment using the GST-Cav
GST plus Cav-1 61-81 (GST neg. ctrl) and
61-101 with or without the F92A
GST plus Cav-1 61-81 fused to a 20 alanine
substitution in combination with endothelial
inactivation mutant (neg ctrl; Fig. 1B). Pull-
cell lysates (allowing the formation of large,
down assays were performed with
native protein complexes) or purified
recombinant eNOS to characterize the
recombinant eNOS (direct protein-protein
eNOS/GST-CAV interaction in absence of
interaction) as previously published by our
confounding endogenous Cav-1 complexes
group. Coating of gluthatione resin with
(see above) and the eNOS used was shown
GST alone, GST-CAV (61-101) or the GST-
to undergo myristoylation, palmitoylation
CAV (61-101) F92A mutant followed by and phosphorylation47. We found that CAV-
incubation with EC lysate or recombinant
A did not increase eNOS binding compared
eNOS resulted in nearly-identical eNOS
to our negative control GST (Fig 1C,D),
binding to CAV WT and CAV F92A (Fig.
whereas with CAV-AB, approximately half
1A and data not shown). Anti-GST
the binding associated with CAV was
WT

  5
recovered (50.2%; Fig. 1C,D). Interestingly, minimal and near-complete recovery of
pull-down with CAV-BC resulted in 92% of eNOS binding when CAV-C aa 96-97 and
eNOS binding compared with our CAV 96-99 are added to CAV-B, respectively
WT
positive control (P = 0.015; Fig. 1C, D) (Fig. 2D), indicating that the bulk of CAV-C
indicating that the bulk of eNOS binding binding to eNOS is mediated by aa 98-99.
(92%) occurs within the CAV-BC region. In Hence, we conclude that CAV-A, F89,
contrast, the two neg. control constructs Y100 and R101 are outside of the eNOS
lacking the intact CAV (81-101) domain binding domain of CAV, which is mediated
(GST and ALA; Fig. 1B) showed <10% within CAV aa 90-99. This was further
eNOS binding compared to wild-type GST- confirmed by the lack or minimal loss of
CAV confirming specific eNOS interaction eNOS binding when R101 or Y100/R101
with CAV and CAV 89-101 (data not CAV-BC mutants were used to pulldown
WT
shown). eNOS (Fig. 2EF).
To determine the individual
importance of CAV-B vs CAV-C, we Binding subdomain and relative K of
d
performed binding experiments with only CAV derived peptides. To gain knowledge
either the CAV-B or -C segments being left on the eNOS binding subdomain of CAV, D
o
w
intact, while the remainder of CAV was which based on our pulldown data (Figs. 1- n
lo
a
inactivated with a series of alanines. 2) is highly restricted to the CAV 90-99 d
e
d
Surprisingly, we found that neither CAV-B section (summarised in Fig. 3A) and fro
m
nhoalrf -oCf stehgem eeNnOtsS, abloinndei,n cgo u(2ld7 %ac caonudn t3 9f%or tdheet eermNiOnSe /CthAe Vd isinsotecriaactitoionn caonnds taitnst s(pKedc)i foicf http://w
respectively of CAV ; Fig. 1D and 1E). binding site, fluorescence polarization w
WT w
This suggests that the eNOS binding site experiments were performed. Fluorescein- .jb
c
was partially located within both CAV-BC labelled CAV peptides and purified .org
sub-regions. recombinant eNOS were used, the latter’s b/
y
g
size expected to increase polarization of u
e
s
eNOS predominantly interacts with labelled peptides following positive t o
n
A
residues 90-99 of CAV. To further identify interaction. CAV-specific interaction was p
the eNOS binding site located within CAV- initially confirmed using a CAV 82-101 ril 5
, 2
BC, we first chose to methodically peptide linked to fluorescein using a charged 0
1
9
reconstitute sections of CAV-B (89-95) to solubility linker from the Cav-1 sequence
the intact CAV-C sub-domain and (EGTHSF; aa 76-81; fluo-CAV), whereas a
accordingly recover eNOS binding to the negative control peptide encoding for the
level of our two positive controls, CAV-BC fluorophore-tagged linker alone was use as
and CAV (Fig. 2A). Gradual addition of negative control (fluo-linker). We have
WT
CAV-B aa 93-95, 91-95 and 90-95 to CAV- observed that increasing concentrations of
C induced cumulative recovery in eNOS eNOS (0-1uM) in combination with a fixed
binding by 19, 23 and 82%, respectively concentration of fluo-CAV (100nM) caused
over CAV-C alone, indicating that a.a. 89 a concentration-dependent appreciation of
was not essential for eNOS binding (Fig. fluorescence polarization. In contrast, fluo-
2AB) since no significant differences was linker peptide exhibited little to no increase
observed between CAV-C+B and CAV- in fluorescence polarization to increasing
90-95
BC. The complement experiment, with concentrations of eNOS (Fig. 3B). Next, we
intact CAV-B followed by gradual compared CAV binding to our herein
restitution of CAV-C (Fig. 2C), resulted in identified eNOS binding subdomain CAV

  6
90-99 and CAV 90-99/F92A. Subtraction of programs used, with a similar orientation
non-specific fluo-linker fluorescence and position in the eNOS (blue) active site
polarization values revealed very similar in close proximity to the iron-binding heme
fluorescence, or binding to eNOS, between moiety (red; Fig. 4B and inset) at the core of
all three peptides studied (Fig. 3C), with Kd the protein rather than at its surface, which
values for CAV (49 nM), CAV 90-99 (42 is directly accessible through the eNOS
nM) and CAV 90-99 F92A (23 nM). groove (Fig. 4B) 15 48. Additional docked
Stoichiometry of the interaction was conformations data show of the F92 residue
estimated by the Hill coefficient and (yellow arrow; Fig. 4C) deep into the
revealed that eNOS/Cav peptides bind hydrophobic pocket of eNOS binding site
roughly between a 1:1 to 1:2 ratio (1.02, (red area, surface representation), whereas
2.13 and 1.4 for CAV 90-99, CAV 90-99 the F92A mutant remains outside this pocket
F92A, and CAV respectively), which may (purple). A more detailed analysis of the
be a result of the slight oligomerization F92 - eNOS hydrophobic pocket interaction
tendencies of the sequence. reveals that F92 makes hydrophobic and π-π
interactions in the eNOS hydrophobic core
CAV-derived peptides interact with eNOS comprising Trp445, Tyr475, Cys184 and Do
w
pocket. In order to better understand the especially Trp447 (Fig. 4D, simplified view nlo
a
CAV-eNOS interaction, a three dimensional and inset). A ball and stick snapshot of the de
d
crystal structure of the WT and F92A CAV F/A92 versions of the peptide is provided fro
m
binding sub-domain was modelled based on (Fig. 4E) and shows how close eNOS h
their high amino acid homology (75%) to Trp447 (pink) is to F92 and making pi-pi ttp://w
the crystal structure of NmrA-like family interactions, while the A92 mutant is distant w
w
domain containing protein 1 and energy with no bond. .jb
c
minimized. Fig. 4A shows the structure with .org
hydrophobic surface representation of WT F92A CAV 90-99 can promote NO release by/
g
(90-TTFTVTKYWF-99) and F92A CAV in an eNOS-specific manner. Since we ue
s
90-99 peptides, clearly showing high herein identify the eNOS binding subdomain t o
n
A
hydrophobicity (red color) around the F92 of Cav-1, we tested whether F92A CAV 90- p
and V94 residues, and the high 99 can behave similarly to F92A Cav-1 and ril 5
, 2
hydrophilicity of the K96 residue (blue). increase NO release. For this purpose, we 01
9
Interestingly, the F92A substitution (Fig. used synthetic, antennapedia-fused peptides,
4A, right) caused little change in the tri- which allow intracellular delivery of cargo
dimensional structure and hydrophobicity peptides. Incubation of cells with an AP-
(reddish color), helping rationalize the fused CAV 90-99 F92A peptide (AP-
similar binding of eNOS to WT and F92A CAV92) at 5 and 10 uM (10uM shown)
Cav-1, increased both nitrate and nitrite release, the
Having knowledge of theoretical stable breakdown products of NO, compared
CAV 90-99 structure, molecular docking with vehicle or control AP peptide (Fig. 5A,
simulations were undertaken using the *P<0.05). We found that NO production
crystal structure of human eNOS as the was NOS inhibitor L-NAME and eNOS
receptor for molecular docking studies. Our siRNA sensitive (data not shown), indicating
data show a very similar docking pattern for that it was eNOS-derived. Subsequently, use
both WT (purple) and F92A CAV 90-99 of an eNOS reconstituted system with low
(green) peptides, which was highly endogenous Cav-1 (eNOS transfected COS-
redundant between the two simulation 7 cells)35 showed that the cell permeable

  7
CAV 90-99 F92A peptide has little effect on to F92A Cav-1, which could help the
basal (12h) NO accumulation. In contrast, management of diseases characterized by
when eNOS is almost completely inhibited abnormally low levels of protective NO
by adenoviral delivery of Cav-1, AP CAV release.
90-99 F92A can promote a sustained NO
release (Fig. 5B). The increased ionophore The Caveolin binding motif and eNOS
NO release shows that eNOS activity was The pioneering observations that eNOS can
never saturated. These data indicated that form a stable complex with Cav-1, causing
our peptide requires eNOS/Cav-1 attenuated NO release and changes in
interaction. Finally, the NO being released cardiovascular homeostasis, where based on
following AP-CAV 90-99 F92A stimulation co-immunoprecipitations, GST pull-downs,
(10uM) is bioavailable, as it can decrease by cotransfections and yeast two-hybrid
55% PAF-induced (100nM, 1h) neutrophil experiments 18, 19. For our experiments, most
adhesion, an effect not observed with our of our GST pull-down assays were
AP vehicle peptide (Fig. 5C, quantification performed using recombinant eNOS, which
D). Finally, we observed that l-NAME allows for the assessment of direct
pretreatment of EC blocked the anti- interaction between eNOS and the D
o
w
adhesion effect of our peptide (data not scaffolding domain, without contributions n
lo
a
shown), directly confirming the implication from cellular protein complexes and by- d
e
d
of NO bioavailability in this therapeutically- products, such as HSP90 and calmodulin, fro
m
relevant effect. two known eNOS / Cav-1 binding h
modulators 19, 21. To err on the side of ttp://w
caution, while we have demonstrated that w
w
DISCUSSION CAV inactivation by alanines completely .jb
c
Recent studies have highlighted the blocks binding to eNOS, stretches of .org
significance of Cav-1-dependent control alanines could form alpha helixes, although b/
y
eNOS both in vitro and in vivo 18, 39, 49, modeling of the scaffolding domain has gu
e
s
stressing the tight relationship between these suggested that CAV itself takes on a alpha t o
n
two proteins. Our latest publication helical structure 51, 52; hence, an induced Ap
highlighted the capabilities of the F92A alpha helical structure via alanines may ril 5
, 2
Cav-1 mutant to promote basal eNOS- unintentionally provide a better than 0
1
9
derived NO release 39, which highlights a expected platform; moreover, others have
novel way to increase biologically active shown that alanine substitution is a reliable
unstimulated NO levels, a critical factor in a approach for dissociating client protein
range of diseases associated with decreased binding to CAV 53.
endothelial function 50. Herein, we provide a With the above-described technique,
molecular characterization of how eNOS the eNOS-binding motif of CAV identified
binds to Cav-1 and its F92A mutant by herein (90TTXTVTKYWF; Fig. 5C)
identifying a short CAV sequence that has overlaps with the ‘caveolin binding motif’ of
retained most of its eNOS binding affinity. Cav-1 (92FTVTKYWFY) 53. The caveolin
Molecular modeling data revealed how binding motif, φXφXXXXφXXφ (where φ
eNOS docks to Cav-1 and cell culture can be Trp, Phe or Tyr), identified using a
experiments show that a non-inhibitory phage library 53 was demonstrated to be
CAV 90-99 peptide can be used as a basis important for Cav-1 interaction with client
for a pharmacophore to promote increased proteins. The removal of aromatic residues
eNOS-derived NO bioavailability, similarly from the caveolin binding motif of a G -
i2
α

  8
derived peptide prevented the peptide from region in Cav-2 differed from Cav-1 by 5
associating with Cav-1 53. Similarly, alanine residues – chiefly the replacement of
substitution of the aromatic residues (in T90/91/93 with alanine, leucine and
bold) within the Cav-1 binding motif of glutamic acid respectively, in addition to
eNOS, 350FSAAPFSGW358, prevented the W97 with valine and F98 with methionine.
inhibition of eNOS activity by Cav-1 15. This strongly supports the usage of the
Interestingly, in contrast to the phage library identified sequence as a basis for a NO
study 53, we found that less than 10 residues release-inducing pharmacophore derived
could account for the majority of eNOS from the F92A Cav-1 sequence, as the
binding, as opposed to the 16 residue regulatory role of CAV appears to be
sequence identified prior, suggesting that the associated specifically with an intrinsic
other residues within the CSD may property of the identified binding sequence.
contribute to specific client protein
interactions. To further support this, our Localization of CAV and F92 in the eNOS
polarization data indicate that the catalytic site:
dissociation constant for the CAV-eNOS A recent review article 57 has suggested that
interaction was not significantly altered the Cav-1 binding site within eNOS is D
o
w
following truncation of CAV. In contrast, it inaccessible short of detrimental or drastic n
lo
a
is of interest to note that others have shown conformational changes to the eNOS d
e
d
that CAV 90-101 mediates less than 5% of protein. However, this is unlikely to be the fro
CinAdiVca t8in2g- 10p1r ofboinudnidn gh etote rGog-penroetietyin iGn i2αthBe, tchaes es, tausd yd,e mceolnlss trthataetd diind an porti oern dstougdeyn o15u. sIlyn http://wm
way Cav-1 docks to its multiple client express eNOS were transfected with an w
w
proteins 54. active mutant of eNOS bearing alanine .jb
c
Taking our data into the context of substitution in lieu of the aromatic residues .org
the multiple Caveolin isoforms, it has been in the eNOS Cav-1 binding motif, b/
y
demonstrated that both Cav-1 and Cav-3, a 350FSAAPFSGW358, which prevented gu
e
s
skeletal muscle specific form of caveolin, inhibition by Cav-1 and therefore t o
n
can interact with eNOS 55; indeed, confirming that the Cav-1 binding motif Ap
introduction of a peptide encoding the Cav-1 within eNOS is indeed accessible to Cav-1. ril 5
, 2
scaffolding domain CAV, or the analogous Cav-1 binding to eNOS is known to 0
1
9
sequence from Cav-3, was sufficient to be ‘antagonized’ by CaM, and vice-versa,
inhibit eNOS activity, whereas the Cav-2 through a complex allosteric mechanism
CAV peptide could not produce this effect involving separate eNOS domains, including
15. This suggested that there should be the CaM binding domain58, 59. Our data
homologous regions between Cav-1 and describe a homology-based crystal structure
Cav-3 that are essential for eNOS binding of CAV 90-99 and its F92A variant, and
and inhibition that would not be present in show stable insertion into the eNOS groove
Cav-2. Interestingly, when comparing the of the oxygenase domain, similar to what
CAV sequences (Fig. 5C) of Cav-1, Cav-2 has been suggested by biochemical
and Cav-3 15, 56 , it was found that there was experiments58-60. Most importantly, the main
a high degree of similarity (90%) between difference between F92 and A92 variants of
the putative eNOS-binding site in Cav-1 and CAV 90-99 comes from the observation that
the analogous region in Cav-3, with only a the F92 forms bonds with eNOS Trp447 and
one amino acid residue substation at position Tyr445 while remaining in close proximity
99 (C in lieu of F); in contrast, the analogous with other key eNOS active site amino

  9
acids, such as Cys184, Met358 and
Ala44661, 62. Since Trp447 is in close
contact with both BH4 and heme, whereas
Tyr475, Ala446 and Cys184 bind heme and
Trp445 and Met358 interact with BH462, an
essential eNOS co-factor for higher eNOS
spin state, our data suggest that Cav-1 F92
residue interferes with BH4’s eNOS
interaction, resulting in eNOS inhibition. To
err of the side of caution, however,
competitive inhibition of Cav-1/eNOS
binding with excess BH4 has not been
verified.
In summary, we have identified a
biologically active 10 residue sequence from
CAV that is capable of promoting eNOS-
derived NO release in endothelial cells. D
o
w
Modeling results suggests that these n
lo
a
peptides potentially bind deep within the d
e
d
Cav-1 binding motif, mimicking a known fro
m
eNOS inhibitor. Furthermore, there is h
significant potential for use of this sequence ttp://w
as the basis for a pharmacophore for w
w
regulating NO activity, as it appears to be an .jb
c
intrinsic property of the sequence. .org
b/
y
g
ACKNOWLEDGEMENTS u
e
s
We would like to acknowledge CIHR, t o
n
A
Heart and Stroke Foundation of Canada, p
British Columbia & Yukon (AT, PB) ril 5
, 2
Canada Foundation for Innovation, British 0
1
9
Columbia Knowledge Development Fund
and NSERC (AS) for salary support. LR
would like to acknowledge support from
NIH grant GM052419.

  10