(Received for publication, September 26, 1995; and in revised form, December 5, 1995)
From the
The calmodulin (CaM)-binding regions in bovine endothelial
nitric oxide synthase (eNOS) and murine inducible nitric oxide synthase
(iNOS) are identified in this study as eNOS residues 493-512 and
iNOS residues 501-532. Peptides corresponding to eNOS
493-512 and iNOS 501-532 produce a
Ca-dependent, electrophoretic mobility shift of CaM
on 4 M urea gels. The two peptides are also potent inhibitors
of the CaM-mediated activation of neuronal nitric oxide synthase and
have dissociation constants for CaM binding of 4.0 and 1.5 nM,
respectively. Substitution of eNOS and iNOS CaM-binding domains in
eNOS/iNOS chimeric proteins produces major alterations in the
Ca
and CaM dependence of the intact enzymes expressed
and purified from a baculovirus/Sf9 insect cell system. Replacement of
aligned iNOS sequence with eNOS 493-512 creates a functional,
chimeric iNOS that is both Ca
- and CaM-dependent.
Replacement of aligned eNOS sequence with iNOS 501-532 creates a
functional, chimeric eNOS that is CaM-independent but that remains
Ca
-dependent. Specific amino acid residues critical
for CaM binding by eNOS are also identified in this study as Phe-498,
Lys-499, and Leu-511 in the bovine eNOS sequence.
Nitric oxide (NO) ()is produced in various tissues by
the oxidation of L-arginine in a reaction catalyzed by the
enzyme NO synthase (NOS). Three distinct isoforms of NOS have been
identified by cDNA cloning and sequencing. NOS cDNAs for the three
isoforms derived from brain (1) ,
macrophage(2, 3, 4) , liver(5) , and
endothelial cells (6, 7, 8, 9, 10) show about
50% identity in deduced amino acid sequence. Each of the sequences
contains conserved regions that correspond to functional domains for
binding of heme, calmodulin (CaM), FMN, FAD, and NADPH (11) .
Type I neuronal NOS (nNOS) and Type III endothelial NOS (eNOS) are
distinguished from Type II inducible NOS (iNOS) in that both are
Ca
-dependent, constitutive enzymes. nNOS and eNOS are
activated in neurons, endothelial cells, and certain other cell types
by agonist-stimulated elevation of intracellular Ca
and resultant binding of Ca
/CaM. In contrast,
NO production by iNOS is thought to be
Ca
-independent. iNOS is induced in macrophages, as
well as in other cells such as hepatocytes (12) and vascular
smooth muscle (13) , in response to cytokines and bacterial
endotoxin. The enzyme is tonically active once induced. However,
despite the fact that it is a Ca
-independent enzyme,
iNOS contains a CaM-binding domain and CaM functions as subunit of the
enzyme, remaining tightly bound even in the absence of elevated
intracellular Ca
(14) . Divergent sequences in
the CaM-binding domains of the constitutive and inducible NOS isoforms,
therefore, may produce major differences in the enzymes in terms of
both their Ca
-dependence and reversibility of CaM
binding.
High affinity CaM-binding domains thus far identified have
very limited degrees of sequence homology. These domains do, however,
possess a number of common structural features. CaM-binding regions are
typically contained within a short stretch of 15-20 contiguous
amino acids that form a basic, amphiphilic -helix within the
target protein. Positively charged residues and hydrophobic residues
are sequestered on opposite sides of the helix(15) . A sequence
with these characteristics has been identified in nNOS (residues
725-747 of the rat cerebellar sequence) as the CaM-binding region
of this enzyme(16, 17) . It has therefore been assumed
that homologous sequences within eNOS and iNOS represent the
CaM-binding domains of these enzymes(11) . eNOS and iNOS
CaM-binding domains, however, have never been definitively identified
or characterized. Furthermore, the CaM-binding domain of iNOS may be
atypical as has been described for certain other proteins that bind CaM
in a Ca
-independent manner. The CaM-binding domain of
the
-subunit of phosphorylase kinase, for example, consists of two
closely adjacent but noncontiguous subdomains(18) . The
presence of two separated CaM-binding domains within the same
polypeptide has also been reported for adenylate cyclase from Bordetella pertussis(19) .
In the present study,
we have experimentally defined and characterized the CaM-binding
domains of eNOS and iNOS. Studies with synthetic peptides and with
eNOS/iNOS chimeric proteins provide insight into the molecular basis
for differences in the Ca and CaM dependence of these
two important enzymes.
where [P] is the total
concentration of added peptide (20 nM) and [CaM] and K are concentrations of calmodulin required to reach
half-maximal activation in the presence and the absence of peptide,
respectively.
Depending on the charge and hydrophobicity
of the peptide, high affinity binding of the peptide to CaM is detected
by this method as a band with increased or decreased mobility relative
to the unbound CaM band. In the presence of Ca, the
electrophoretic mobility of CaM was retarded by a peptide corresponding
to bovine eNOS residues 493-512 (Fig. 1A),
suggesting that this sequence (identified previously by Lamas et
al.(6) as the eNOS CaM-binding domain) contains all of
the residues necessary for high affinity binding of
Ca
/CaM by eNOS. At a 2:1 ratio of CaM to peptide,
half of the CaM formed a CaM-peptide complex. At a 1:1 ratio of CaM to
peptide, all of the CaM was gel shifted, indicating a 1:1 stoichiometry
of peptide-CaM complex formation. The eNOS 493-512 peptide,
however, did not form a complex with CaM in the presence of the
Ca
chelator, EGTA, indicating that complex formation
is Ca
-dependent. Similar results were obtained when
electrophoretic mobility shift assays were performed under
nondenaturing conditions, indicating that 4 M urea is not
required to demonstrate peptide-CaM complex formation.
Figure 1:
Electrophoretic mobility shift of CaM
by synthetic peptides. CaM (5 µg) was incubated with the indicated
peptide in the presence of 0.1 mM CaCl or 2 mM EGTA. Samples were then electrophoresed on 4 M urea-15%
polyacrylamide gels containing either 0.1 mM CaCl
or 2 mM EGTA. The relative mobilities of CaM (indicated
by arrows) and CaM-peptide complexes were visualized by
Coomassie staining. Molar ratios of peptide:CaM of 0:1, 0.5:1, 1:1, and
2:1 are indicated. The results are representative of three separate
experiments.
A synthetic
peptide from iNOS that represents those residues in the murine iNOS
sequence (501-523) that are aligned with bovine eNOS residues
493-512 (11) was also tested for its ability to bind
either Ca/CaM or the Ca
-deficient
conformation of CaM (apoCaM). No complex formation was detected for the
peptide and apoCaM. Furthermore, only trace amounts of
Ca
/CaM were retarded on the 4 M urea gel
even at a 2:1 molar ratio of peptide to CaM (Fig. 1B).
Thus, it appears that residues 501-523 of murine iNOS (which
completely encompasses the 504-522 sequence identified by
Lowenstein et al.(3) as the iNOS CaM-binding domain)
does not represent the entire sequence necessary for either
Ca
-dependent or Ca
-independent
binding of CaM by iNOS. A longer 501-532 peptide sequence from
murine iNOS (which encompasses the longer 503-532 sequence
suggested by Xie et al.(2) as the iNOS CaM-binding
domain) was also examined for its ability to bind either
Ca
/CaM or apoCaM. In contrast to the shorter sequence
(501-523), the longer sequence (501-532) formed a 1:1
complex with CaM in the presence of Ca
(Fig. 1C). Residues 524-532 in iNOS,
therefore, which are located outside of the iNOS sequence aligned with
the complete eNOS CaM-binding sequence, appear to be necessary for high
affinity binding of CaM by the enzyme. Interestingly, no peptide-CaM
complex formation occurred with the longer peptide in the presence of 2
mM EGTA, indicating that CaM binding (even by the longer iNOS
sequence) is Ca
-dependent.
The
Ca-dependence of CaM binding by the iNOS
501-532 sequence suggests at least three alternative
possibilities with regard to the interaction of CaM with the intact
iNOS enzyme. One possibility is that binding of CaM by iNOS (which
occurs in many cell types under resting cell Ca
levels) is not a truly Ca
-independent event but
requires low concentrations of Ca
. A second
possibility is that binding of CaM by iNOS involves two closely
adjacent subdomains, as has been described previously for the
-subunit of phosphorylase kinase(18) . Binding of CaM to
each individual subdomain in phosphorylase kinase is
Ca
-dependent. However, synergism between the two
adjacent subdomains in the intact protein produces extremely high
affinity for CaM even in the absence of Ca
. If this
type of interaction were to occur between iNOS and CaM, a second
unrecognized CaM-binding domain may exist in iNOS adjacent in the
primary structure to the one predicted by computer
alogorithm(25) . A third possible explanation of the gel shift
data is that regions or domains in iNOS that are not adjacent to the
CaM-binding domain in the protein primary structure (and that may be
physically far removed in the tertiary structure of the macromolecule)
are required to produce a binding interaction that is not dependent on
the presence of Ca
.
The possibility that the murine iNOS sequence contains
a second CaM-binding domain immediately adjacent to that contained
between amino acids 501-532 was also investigated. Two 26-residue
peptides representing murine iNOS sequence immediately amino-terminal
(residues 475-500) and immediately carboxyl-terminal (residues
533-558) to the 501-532 sequence were prepared and tested
for their abilities to inhibit nNOS. No inhibition was observed with
either peptide even at peptide concentrations as high as 1000
nM. Therefore, if a second CaM-binding domain does exist in
iNOS, it appears that it is not located adjacent to the first domain as
in the case, for example, of the two subdomains of the -subunit of
phosphorylase kinase(18) .
Catalytic activity of each of the five purified NOS
enzymes was measured in the presence of varying concentrations of
exogenously added CaM (Fig. 2). Assays were carried out in the
presence of Ca (2.5 mM) and in the absence
of Ca
(achieved by addition of 10 mM EGTA).
Wild-type eNOS, purified from the baculovirus expression system, was
completely dependent on both Ca
and CaM (Fig. 2A). Wild-type iNOS, on the other hand, was
completely CaM-independent as well as significantly (although not
entirely) Ca
-independent (Fig. 2B).
Purified iNOS thus appears to contain irreversibly bound CaM, which
does not dissociate from the enzyme even during purification in the
presence of 2 mM EGTA. Moreover, purified iNOS was
consistently found to be twice as active in the presence of
Ca
than in its absence. Although other investigators
have reported previously that iNOS can be partially inactivated by
EGTA(32, 33, 34) , many researchers currently
assume that iNOS is completely unaffected by Ca
chelation. iNOS activity determinations of tissue homogenates are
thus routinely carried out in the presence of high concentrations of
EGTA. Therefore, we sought to determine whether iNOS isolated from
macrophages displays the same partial Ca
dependence
observed for the baculovirus-expressed enzyme. iNOS was purified from
activated murine RAW 264.7 macrophages by the method used for
purification of the baculovirus-expressed enzyme. iNOS activity of
crude macrophage homogenates and of the purified macrophage-expressed
enzyme was inhibited by 10 mM EGTA to approximately the same
extent observed with the baculovirus-expressed enzyme. These results
suggest that iNOS activity determinations that are performed in the
presence of EGTA may underestimate enzyme activity by up to 2-fold.
Figure 2:
Ca2+ and CaM dependence of wild-type
and chimeric NOS enzymes. NOS activity was determined by estimating the
rate of conversion of L-arginine to L-citrulline.
Assays were performed in duplicate in the presence of varying
concentrations of CaM and in the presence of either 2.5 mM CaCl or 10 mM EGTA. Similar results were
obtained in three different experiments.
Replacement of the CaM-binding sequence of eNOS (residues
493-512) with aligned sequence from iNOS (residues 501-523)
resulted in creation of a chimeric enzyme that had properties distinct
from both wild-type eNOS and wild-type iNOS (Fig. 2C).
The I 501-523 eNOS chimeric enzyme was activated by both apoCaM
and Ca/CaM. The iNOS 501-523 sequence by
itself, however, was not sufficient to produce irreversible
CaM-binding. Thus, I 501-523 eNOS (like wild-type eNOS) remained
dependent on exogenously added CaM for activity. In contrast, the
longer iNOS 501-532 sequence was sufficient to confer CaM
independence on eNOS. I 501-532 eNOS (like wild-type iNOS) was
completely independent of added CaM (Fig. 2D). An
additional conclusion that can be drawn from the CaM-independence
observed for I 501-532 eNOS is that regions in iNOS that are
required for irreversible CaM binding (but that are located outside of
residues 501-532) are also conserved in the eNOS enzyme. If these
other putative domains did not exist in eNOS, I 501-532 eNOS
would be expected to remain CaM-dependent (like wild-type eNOS).
Interestingly, irreversible association of CaM with eNOS (observed with
I 501-532 eNOS) was not sufficient to activate the enzyme in the
absence of Ca
(Fig. 2D). It appears
that bound CaM must be in the Ca
/CaM conformation in
order to interact with eNOS in a manner that activates the enzyme. A
final conclusion apparent from analysis of the chimeric enzymes is that
the 501-523 region of iNOS (which was not sufficient to produce
irreversible CaM binding in eNOS) is necessary for the Ca
and CaM independence of iNOS. This is illustrated by the
endothelial 493-512 iNOS chimeric enzyme, which was completely
dependent on both Ca
and CaM for catalytic activity (Fig. 2E).
The importance of specific hydrophobic and basic residues to eNOS
CaM binding was assessed using two different approaches. Peptides were
prepared in which Phe-498, Val-505, Leu-511, Arg-494, Lys-495, Lys-496,
Lys-499, and Lys-506 were each individually replaced by alanine
residues. Mutated peptides were then compared with the wild-type
peptide for their ability to gel shift CaM on 4 M urea gels as
well as for their inhibitory potency for the CaM-mediated activation of
nNOS. Peptides that were mutated at either Phe-498, Leu-511, or Lys-499
lost their capacity for high affinity binding of CaM, as evidenced by
loss of CaM mobility shift on 4 M urea gels (Table 2).
Furthermore, these same three peptides showed significantly lower
potencies for inhibition of nNOS than did the wild-type peptide. The
hydrophobic residues, Phe-498 and Leu-511, as well as the basic
residue, Lys-499, thus appear to represent essential determinants of
the eNOS/CaM interaction. Phe-498 appears to be especially important
because mutation at this residue results in a 150-fold increase in the
IC of the mutant peptide for inhibition of nNOS.