From the Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446
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ABSTRACT |
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The primary sequences of the three mammalian
nitric- oxide synthase (NOS) isoforms differ by the insertion of a
52-55-amino acid loop into the reductase domains of the endothelial
(eNOS) and neuronal (nNOS), but not inducible (iNOS). On the basis of studies of peptide derivatives as inhibitors of ·NO formation
and calmodulin (CaM) binding (Salerno, J. C., Harris, D. E.,
Irizarry, K., Patel, B., Morales, A. J., Smith, S. M., Martasek, P., Roman, L. J., Masters, B. S., Jones, C. L., Weissman, B. A., Lane, P., Liu, Q., and Gross, S. S. (1997) J. Biol. Chem. 272, 29769-29777), the insert
has been proposed to be an autoinhibitory element. We have examined the
role of the insert in its native protein context by deleting the insert
from both wild-type eNOS and from chimeras obtained by swapping the
reductase domains of the three NOS isoforms. The Ca2+
concentrations required to activate the enzymes decrease significantly when the insert is deleted, consistent with suppression of
autoinhibition. Furthermore, removal of the insert greatly enhances the
maximal activity of wild-type eNOS, the least active of the three
isoforms. Despite the correlation between reductase and overall
enzymatic activity for the wild-type and chimeric NOS proteins, the
loop-free eNOS still requires CaM to synthesize ·NO. However,
the reductive activity of the CaM-free, loop-deleted eNOS is enhanced
significantly over that of CaM-free wild-type eNOS and approaches the
same level as that of CaM-bound wild-type eNOS. Thus, the inhibitory
effect of the loop on both the eNOS reductase and
·NO-synthesizing activities may have an origin distinct from the loop's inhibitory effects on the binding of CaM and the concomitant activation of the reductase and ·NO-synthesizing activities. The
eNOS insert not only inhibits activation of the enzyme by CaM but also
contributes to the relatively low overall activity of this NOS isoform.
The enzymatic activities of the three
NOS1 isoforms (1-7) differ
in their Ca2+-dependence: nNOS (NOS-I) and eNOS (NOS-III)
are Ca2+-dependent constitutive isoforms,
whereas iNOS (NOS-II), as typified by the inducible macrophage and
hepatocyte form, is essentially Ca2+-independent. This
difference in the Ca2+ dependence of the NOS isoforms is
the result of a Ca2+ requirement for the reversible binding
of CaM to the constitutive isoforms (8, 9), in contrast to the almost
Ca2+-independent, high affinity binding of CaM to the
inducible isoform (10).
The NOS isoforms are also differentiated by their maximum enzymatic
activity, nNOS (8, 11-14) and iNOS (15-18) exhibiting much higher
overall activities than eNOS (19-24). We have shown that the lower
activity of eNOS is caused by a lower ability of its flavoprotein
reductase domain to transfer electrons to the catalytic heme domain
(25). However, the structural features that impair the reductase
activity in eNOS, and hence lower the overall catalytic activity,
remain unknown.
Recent evidence (26) suggests that the Ca2+ dependence of
nNOS and eNOS is caused by the presence of an autoinhibitory loop, absent in iNOS and P450 reductase, which interferes with the binding of
CaM (Fig. 1). The presence of such an
autoinhibitory moiety has precedence among CaM-binding enzymes. Indeed,
the skeletal and smooth muscle myosin light chain kinases,
multifunctional CaM-dependent protein kinase II,
CaM-dependent protein phosphatase 2B, calcineurin, and
phosphorylase kinase are among the CaM-dependent enzymes
that possess autoinhibitory structural elements (27). In these enzymes,
CaM acts by displacing an autoinhibitory element that interferes with
substrate access to the catalytic domain. In calcineurin, the
autoinhibitory element is composed of two
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helices of 23 amino acids
located 54 amino acids after the CaM binding sequence (28, 29). In nNOS
and eNOS, the proposed autoinhibitory domain is composed of a
52-55-amino acid insert within the FMN binding domain located
approximately 80 amino acid residues after the CaM binding sequence
(Fig. 1). Location of the putative autoinhibitory element near the
junction between the NOS oxygenase and reductase domains is consistent
with a possible role in controlling electron transfer between these two
domains.
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Fig. 1.
Sequence alignment of various NOS isoforms
and P450 reductase. Homology as was determined using the Pileup
program in the GCG suite of sequence analysis programs. For NOS
alignments, GAP creation penalty = 12, GAP extension penalty = 4. For NOS/CPR, values were 6 and 2, respectively. Black
highlighting in NOS sequences indicates residues identical among
all NOS isoforms. Black highlighting in the P450 reductase
sequence indicates P450 reductase residues identical to these conserved
NOS residues. Gray highlighting indicates the residues
retained in the loop deletion mutants. dm-cnos, Drosophila
melanogaster constitutive
Ca2+/CaM-dependent NOS, GenBank accession
U25117 (47). as-inos, Anopheles stephensi (mosquito)
parasitic protozoan-induced NOS, GenBank accession AF053344 (48).
rp-cnos, Rhodnius prolixus (blood-sucking insect) NOS,
GenBank accession U59389 (49). h-enos, Homo sapiens
constitutive endothelial NOS, GenBank accession M93718 (50). b-enos,
Bos taurus (bovine) constitutive endothelial NOS, GenBank
accession M95674 (51). h-nnos, H. sapiens constitutive brain
NOS, GenBank accession L02881 (52). oc-nnos, Oryctolagus
cuniculus (rabbit) constitutive brain NOS, GenBank accession
U91584 (Y. Jeong and J. Yim, unpublished direct submission to GenBank).
rn-nnos, Rattus norvegicus (rat) constitutive brain NOS,
GenBank accession X59949 (54). h-inos, H. sapiens
cytokine-induced colorectal adenocarcinoma NOS, GenBank accession
L24553 (55). cp-inos, Cavia porcellus (guinea pig) inducible
lung NOS, GenBank accession AF027180 (59). mm-inos, Mus
musculus (mouse) inducible macrophage NOS, GenBank accession
M87039 (57). gg-inos, Gallus gallus (chicken) inducible
macrophage NOS, GenBank accession U46504 (58). om-inos,
Oncorhynchus mykiss (trout) inducible macrophage NOS, from the
partial sequence in GenBank accession X97013 (53). h-cpr cytochrome
P-450 reductase, H. sapiens liver, SWISS-PROT accession
P16435 (56).
To establish definitively whether the peptide insert in its native
protein context functions as an autoinhibitory loop, we have
constructed mutants of wild-type and chimeric NOS enzymes lacking the
insert and have studied the consequences of deleting the loop on their
Ca2+ dependence and catalytic activities. Our results
clearly identify the eNOS insert as an autoinhibitory loop that
functions not only as an effector of the Ca2+ dependence
but also as an electron transfer control element that lowers the
catalytic activity of eNOS relative to that of nNOS and iNOS.
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EXPERIMENTAL PROCEDURES |
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Materials-- Bovine endothelial NOS expression plasmid 6xHis-pcWori/beNOS was identical to that described previously (20). pBluescript/iNOS was provided by Steve Black (University of California, San Francisco). The mouse macrophage NOS and human CaM expression plasmids were constructed as reported previously (18, 30). Enzymes used in DNA manipulation were from New England Biolabs (Beverly, MA). L-Arg was from Aldrich, (6R)-5,6,7,8-tetrahydrobiopterin from Alexis Biochemicals (San Diego), and HEPES buffer from Fisher. Recombinant human CaM was purified from Escherichia coli according to published procedures (31). DNA purification kits and Ni2+-nitrilotriacetic acid-agarose were purchased from QIAGEN (Chatsworth, CA). BL21(DE3)-competent cells were from Novagen (Madison, WI). All other reagents and materials were from Sigma.
DNA Manipulations--
The genes encoding the N/E and I/E
chimeras were prepared as described previously (25). The structures of
all of the protein constructs employed in this study are shown
schematically in Fig. 2. All PCR
extensions were performed with VentR polymerase, which
possesses a 3'-5' proofreading exonuclease to minimize extension
errors. The E portion was generated by PCR primer extension using
template pBluescript/eNOS, primer 1 (5'-CAACC ATCCT GTACG
CTAGC GAGAC CGGCC GGG), which introduced the 5'-NheI
splice site (in bold), and primer 2 (5'-CAGCC CCTCT CTTCT AGAAC TGCAG TGG), which produced a 3' XbaI site (bold)
after the stop codon. These sites were used to produce pcWori//N/E by subcloning of the eNOS reductase domain PCR fragment into
pcWori/nNOSNheI761, a Thr761 Ser
nNOS mutant possessing the NheI splice site (25). Ser is the
homologous amino acid found in eNOS. The iNOS fragment containing the
heme domain and CaM binding of I/E was generated by PCR extension using
template pBluescript/iNOS and primers 3 (5'-AGTCT CACAT
ATGGC TTGCC CGTGC AAGTT TCTGT TCAA) and 4 (5'-CGGGC GTCGC
TAGCA AAGAG GACTG TGGC) to produce, respectively, 5'-NdeI and 3'-NheI restriction sites (bold),
which allowed for subcloning into pcWori//N/E.
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The genes encoding for chimeras E/I and N/I were prepared in a similar manner. The iNOS reductase domain was generated by PCR primer extension using primer 5 (5'-CACAT GCCTC TTTGC TAGCG AGACA GGGAA GTCT) and primer 6 (5'-TGCTC TAGAT CAGAG CCTCG TGGCT TT) to produce NheI and XbaI sites (bold), respectively. These sites were used to subclone the macrophage reductase domain PCR fragment into pcWori//E/N and pcWori//N/E, producing pcWori//E/I and pcWori//N/I, respectively.
The eNOS loop deletion was made by overlap extension PCR mutagenesis
(32) using template pcWori//N/E and primers 7-10 (5'-ATTAA CTAGT CCCGT
CCTTT GAATA CCAG, 5'-GGTGC CCAGG GCGCC TGCAC TCTCC ATCAG, 5'-ATGGA
GAGTG CAGGC GCCCT GGGCA CCCTC, and 5'-CAGCA GCTGG AGGCC C). Two PCR
fragments were generated using primers 7 (which anneals before the
NheI site) and 8 (mutagenesis primer), producing fragment A;
and primers 9 (mutagenesis primer) and 10 (which anneals 20 bases after
the unique KpnI site of eNOS), producing fragment B. A and B
possessed a 15-base overlap, allowing for mutually primed synthesis of
full-length AB using A, B, primer 7, and primer 10. Subcloning via the
5'-NheI and 3'-KpnI sites into pcWori//N/E yielded pcWori//N/E. Sequencing was performed to ensure against PCR
errors in fragment AB. To produce pcWori//E/E
, the parent chimera
E/E plasmid, whose splice site produces a silent mutation, was produced
by subcloning via NheI and XbaI the E reductase
domain fragment from pcWori//N/E into pcWori//E/N followed by
subcloning of the above E
fragment (AB, digested with
NheI and KpnI) into pcWori//E/E.
Protein Expression and Purification-- Expression and purification were performed as described previously (25).
Activity Assays--
The rate of ·NO synthesis,
determined from the oxidation of oxyhemoglobin to methemoglobin, and
that of cytochrome c reduction were measured at 37 °C as
reported previously (25). Where specifically indicated, an additional
100 mM KCl was included in the incubation mixture.
Ferricyanide reduction was monitored at 420 nm using an extinction
coefficient of 1.2 mM1 cm
1 with
an assay solution identical to that used for the other assays except
that potassium ferricyanide was added to a final concentration of 0.8 mM in place of cytochrome c or oxyhemoglobin.
Protein concentrations were estimated using an extinction coefficient
of
Soret = 100 mM
1
cm
1 for the NOS proteins and
276 nm = 3300 M
1 cm
1 for human CaM.
Calcium Dependence Measurements--
Free calcium concentrations
were reproduced as reported before (33) at an ionic strength of 100 mM KCl (or NaCl) and using the value
KD (Ca2+-EGTA) = 27.9 nM
at pH 7.50, 37 °C.
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RESULTS |
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Expression and Purification-- Expression and purification of the NOS proteins (for protein structures, see Fig. 2) was done as reported previously using Ni2+-nitrilotriacetic acid-agarose and 2',5'-ADP-agarose affinity chromatography (25). The final purity of all of the proteins was judged to be greater than 95% by SDS-polyacrylamide gel electrophoresis, and the spectra of all the proteins were consistent with the formation of heme-bound, functional proteins (not shown).
Ca2+ Dependence--
The insert in the eNOS and nNOS
reductase domains may function as an autoinhibitory element that
impedes the Ca2+-dependent binding of CaM
except at high Ca2+ concentrations (26). We therefore
examined the ·NO synthesizing activity of the enzymes as a
function of the free-Ca2+ concentration. The effects of the
insert in the reductase domain on the Ca2+ and CaM
dependence of the activities of the recombinant proteins were measured
in the presence of 500 nM CaM and various free
Ca2+ concentrations (Fig. 3).
The desired free Ca2+ concentrations were obtained by
adding EGTA and Ca2+-EGTA as indicated by the
Ca2+-EGTA equilibrium dissociation constant at the given
temperature and ionic strength (33). The ionic strength in these
studies was controlled by adding KCl to the buffer. The results thus
obtained with wild-type nNOS (Fig. 3A), from which an
apparent KD (KD(app)) of
300 nM for Ca2+ is calculated, were consistent
with those reported by Ruan et al. (34). A slightly lower
value of 150 nM was obtained for eNOS. The iNOS activity
was independent of the free Ca2+ concentration, again in
accord with Ruan et al. (34). The
KD(app) values for Ca2+
in the N/E, E/I, and N/I chimeras were 200, 10, and 10 nM,
respectively (Fig. 3, A and B). For these three
chimeras, the Ca2+ dependence correlates well with the
presence (N/E) or absence (E/I, N/I) of the insert in the reductase
domain.
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Proteins with the insert-deleted eNOS reductase domain required
significantly less free Ca2+ for catalytic activity than
their parent isoforms (Fig. 3B). eNOS, which only differs
from wild-type eNOS by deletion of the putative autoinhibitory loop,
had a KD(app) value of 20 nM
for Ca2+ rather than 150 nM and
thus was activated by a 7-fold lower Ca2+ concentration.
Likewise, activation of N/E
required 4-fold less Ca2+
(50 nM) than the parent N/E chimera (200 nM).
The lower KD(app) values for Ca2+ in
the proteins without the insert approach those for the E/I and N/I
chimeras, which bear the insert-less iNOS reductase domain (e.g. compare the open circles and open
squares in Fig. 3A with the open diamonds
and open squares in Fig. 3B). The I/E and I/E
chimeras did not differ significantly in their Ca2+
dependence; both exhibited some activity even at a 0.1 nM
free Ca2+ concentration, but their activity was not fully
expressed at the lowest Ca2+ concentrations (Fig.
3B).
·NO Synthesis-- NOS-dependent ·NO synthesis, monitored via the oxidation of oxyhemoglobin, and cytochrome c reduction were assayed in two different buffers. The first buffer, which included flavins and other necessary cofactors, was similar in its lack of added salt to buffers conventionally employed to assay the pure proteins (35, 36). The second buffer differed from the first only in that it contained 100 mM KCl to reproduce the conditions employed to assay Ca2+ dependence.
In the presence of CaM, the activity of the wild-type constitutive NOS
isoforms is expected to approach zero when
[Ca2+]free
KD(app) for Ca2+ (Fig.
3A). A similar Ca2+ dependence might be expected
for the mutants without the insert, but the maximum NOS activities for
the insert-less mutants might be expected to be the same as those for
the corresponding CaM-bound wild-type proteins when
[Ca2+]free
KD(app) for Ca2+. Under these
conditions, the CaM-bound insert-less eNOS mutant should have the same
activity as CaM-bound, wild-type eNOS. Contrary to this expectation,
the proteins without the peptide insert have enhanced catalytic
activities (Fig. 4). In the presence of a
high salt concentration, the activities of eNOS
, N/E
, and I/E
(51.4 ± 2.3, 128 ± 19, and 55.4 ± 1.2 min
1, respectively) are approximately double those for
the parent eNOS, N/E, and I/E proteins (23.5 ± 2.3, 69.9 ± 2.0, and 32.6 ± 2.8 min
1, respectively). Identical
effects were seen with KCl or NaCl as the ionic strength buffer (not
shown). Thus, in the presence of an approximately physiological salt
concentration, the loop deletion mutants consistently expressed a
higher activity than the corresponding parent proteins.
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Under low salt conditions, the activities of eNOS and the chimeras
bearing the eNOS reductase domain were comparable; the eNOS, N/E, and
I/E activities were 17.9 ± 1.8, 28.0 ± 2.3, and 16.4 ± 0.7 min1, respectively. Comparison with the high salt
values reported above shows that the eNOS and I/E activities are
increased modestly by salt. However, although the activity of N/E in
the absence of salt was only slightly higher than that of the other
eNOS reductase domain-containing proteins, in the presence of salt, N/E
exhibited an activity (69.9 ± 2.0 min
1) comparable
to the higher activities of the loop-deleted eNOS
and I/E
mutants
(51.4 ± 2.3 and 55.4 ± 1.2 min
1, respectively).
The chimeras were the only proteins whose activities were altered
significantly by changes in the salt concentration, with a higher
activity being observed for chimeras with the eNOS reductase domain and
a lower activity for those with the iNOS reductase domain. Significant
salt-dependent changes in activity were not seen for
wild-type eNOS or for the loop-deleted mutant eNOS in which the
wild-type quaternary structure is presumably preserved better than in
the chimeras. The increases in the activities caused by salt alone are
therefore probably linked to changes in the quaternary structure
introduced by the chimeric construct. The subtle change in the
reductase/oxygenase interface which would suffice to increase electron
transfer and catalytic activity is consistent with the suggestion that
salt effects modulate interdomain electron transfer without causing
major changes in the NOS structure (37).
Cytochrome c Reduction-- Cytochrome c reduction can be used to measure the intrinsic reductase activities of both NOS (38) and P450 reductase (39-41). For eNOS and nNOS, the binding of CaM stimulates cytochrome c reduction by up to 10-fold (38). The cytochrome c-reducing activities of iNOS (18, 25) and CaM-stimulated nNOS (12, 38) are comparable to that of P450 reductase (40-42).
In the presence of CaM and 500 µM Ca2+, the
trends in the cytochrome c reductase activities of the
proteins adhere to the pattern observed for ·NO synthesis. The
rates of the CaM-bound loop deletion mutants (Fig.
5, gray bars) are up to 3-fold
higher than those of the corresponding parent proteins. Thus, the
activities (+KCl) of eNOS, N/E
, and I/E
are 3,440 ± 210, 5,580 ± 210, and 1,990 ± 130 min
1,
respectively, compared with 1,354 ± 24, 1,770 ± 49, and
1,373 ± 90 min
1 for the corresponding parent
proteins.
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The enhancement of cytochrome c reduction caused by removal
of the insert is even more pronounced for the CaM-free proteins (Fig.
5, white bars, and Table I):
CaM-free eNOS and N/E
had activities of 2,150 ± 120 and
2,720 ± 370 min
1, respectively, values that are
10-30-fold higher than those for the corresponding parent proteins
(111 ± 4 and 119 ± 5 min
1, respectively). In
accord with this finding, the E/I and N/I chimeras with the iNOS
loopless reductase have high cytochrome c-reducing
activities in the absence of CaM (3,360 ± 150 and 4,390 ± 170 min
1). The CaM-free activities of I/E and I/E
could not be determined because the proteins had to be coexpressed with
CaM. As for overall NOS activity, the cytochrome c reduction
rates of the proteins with the iNOS reductase suffered a rate decrease
of 50-80% at high salt concentrations.
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CaM Dependence of Cytochrome c Reduction--
A comparison of the
white and gray bars for each protein in Fig. 5
shows that the stimulation ("disinhibition") by CaM is much lower
for the loop-deleted mutants. The binding of CaM to eNOS and N/E, which
have the wild-type eNOS reductase domain, enhances their activities 12- and 15-fold, respectively. In contrast, the binding of CaM to the
corresponding loop-deleted mutants only enhances that of eNOS by a
factor of 0.5 and N/E
by a factor of 2. The comparison could not be
made for I/E and I/E
because the proteins had to be coexpressed with
CaM. The greater enhancement in the cytochrome c reductase
activities of the CaM-free than CaM-bound NOS proteins caused by insert
deletion in each instance makes the activities of the CaM-free and
CaM-bound proteins nearly equal, as indicated by a lowering in the
ratio of the rates (Table I).
The high reductase activities of the CaM-free, insert-deleted proteins
are similar to those of the CaM-free chimeras with the insert-less iNOS
reductase domain. For example, the cytochrome c reduction
rates are comparably high for E/I in the presence and absence of CaM
(3,360 ± 150 and 21,37 ± 27 min1,
respectively), and the corresponding rates for N/I are 4,390 ± 170 and 1,654 ± 27 min
1, respectively. Similarly,
the isolated iNOS reductase domain expressed in E. coli has
high reductase activity (43).
Ferricyanide Reduction--
Ferricyanide reduction provides an
independent and useful measure of reductase activity. Electron transfer
from P450 reductase to ferricyanide occurs from the FAD cofactor, in
contrast to electron transfer to cytochrome c, which occurs
from the FMN cofactor (44). In the case of NOS, the binding of CaM
accelerates electron transfer to both cytochrome c and
ferricyanide (45). We have examined ferricyanide reduction by two
proteins containing the eNOS reductase domain, eNOS and N/E, and by the
corresponding loop deletion mutants, eNOS and N/E
. In the absence
of CaM, the consequences of removing the peptide loop were essentially
the same for ferricyanide reduction as for cytochrome c
reduction (Fig. 6, A and
B, white bars and Table
II). A modest enhancement was observed in
the activities of the proteins when the assays were run at a higher
salt concentration. eNOS and N/E reduced ferricyanide at similar rates
in the low salt medium (482 ± 54 and 955 ± 32 min
1) and gave rise to similar modest increases in
activity when salt was added (939 ± 105 and 2116 ± 230 min
1). Deletion of the inserts in the reductase domains
of these proteins, yielding eNOS
and N/E
, increased
ferricyanide-reducing activity 3-7-fold (to 3,160 ± 120 and
3,180 ± 150, respectively) in the low salt medium and to a larger
extent (5,890 ± 220 and 5,712 ± 220 min
1)
under higher salt conditions.
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The effects of deleting the loops on ferricyanide reduction were much
less dramatic for the CaM-bound than CaM-free proteins (Fig. 6,
A and B, gray bars). In the absence of
salt, CaM-bound eNOS and N/E reduced ferricyanide at similar rates of
2,250 ± 190 and 3,470 ± 230 min1,
respectively. eNOS
and N/E
had higher rates of 8,480 ± 910 and 7,840 ± 400 min
1, respectively. In the presence
of salt, CaM-bound eNOS
had a higher rate than eNOS (13,100 ± 820 min
1 and 6,950 ± 310, respectively), and N/E
had a similarly higher rate than N/E (14,430 ± 500 and 9,850 ± 390 min
1). Thus, although loop-deleted proteins showed
an enhancement in ferricyanide reduction (less than 2-fold in the
presence of salt), the enhancement was much less than the enhancements
in the CaM-free proteins (3-6-fold in the presence of salt). This result agrees with the finding that the loop deletions caused smaller
enhancements of cytochrome c reduction in the CaM-bound than
CaM-free proteins (e.g. in Fig. 4, compare the difference between eNOS minus CaM and eNOS
minus CaM with the difference between eNOS plus CaM and eNOS
plus CaM).
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DISCUSSION |
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The Ca2+ dependence of ·NO synthesis is a major
distinguishing factor among the NOS isoforms. nNOS and eNOS, which have
an insert in the FMN binding domain, have a much higher
Ca2+ requirement than iNOS, which does not have such an
insert. As we show here, deletion of the putative autoinhibitory insert
dramatically lowers the Ca2+ requirement for ·NO
synthesis by both eNOS and the N/E
(Fig. 3B).
Furthermore, replacement of the reductase domains of eNOS and nNOS with
the loopless iNOS reductase domain produces the loopless E/I and N/I chimeras that have a similarly shifted Ca2+ requirement
(Fig. 3A). Thus, all of the NOS proteins without the insert,
whether based on the wild-type or a chimeric structure, have a lower
Ca2+ requirement for activity than the corresponding
insert-bearing parent protein.
In addition to shifting the Ca2+ requirement, deletion of
the loop unexpectedly produces a modest to large enhancement in the maximum rate of ·NO synthesis. Thus, the activities of eNOS,
N/E
, and I/E
are higher than those, respectively, of the parent
eNOS, N/E, and I/E proteins (Fig. 4), the increase being larger in the
presence of a physiological salt concentration. This increase in
·NO synthesis was caused by an increase in the reductase
activity (see below), as found previously for other chimeric proteins
(25).
Three key observations must be addressed regarding the effect of the loop on reductase activity. 1) Removal of the loop yields a protein with a reductase activity approaching that of the corresponding CaM-bound parent protein. 2) CaM is required for ·NO synthesis despite the increased reductase activity of the loop-free proteins. 3) The loopless proteins have a higher reductase activity than the corresponding loop-possessing parent protein whether CaM is bound or not.
The binding of CaM to nNOS has been reported to stimulate electron transfer not only from the flavins to cytochrome c but also from NADPH to the flavins (45). The reduction of ferricyanide and cytochrome c by P450 reductase has been shown to occur at different sites (44). Cytochrome c accepts electrons exclusively from the FMN, whereas ferricyanide accepts electrons from the FAD and possibly also the FMN. Deletion of the loop increases the rates of cytochrome c (Fig. 5) and ferricyanide (Fig. 6) reduction by the NOS proteins in both the CaM-free and CaM-bound states. All of the proteins with a specific loop-deleted reductase domain have similar reductase activities if they are compared either in the CaM-free or CaM-bound state (Fig. 5). Furthermore, the reductase activities of these proteins in the absence of CaM are higher than that of the CaM-bound wild-type reductase (Fig. 5).
CaM enhances cytochrome c and ferricyanide reduction by the
loop deletion mutants less effectively than by the parent proteins (Tables I and II). A restatement of this is that the CaM-free, loop-deleted proteins already reduce cytochrome c and
ferricyanide at rates that more closely approach those for the
CaM-bound proteins. A corollary of this is that loop removal increases
the reductase activities of the CaM-free proteins to a greater extent
than it does the activities of the CaM-bound enzymes. These
differences, which are in accord with the finding that the CaM-free
proteins are the most stimulated when the insert is deleted, establish that the insert in the eNOS and nNOS reductase domains fulfills an
autoinhibitory role. In principle, autoinhibition should be relieved in
the CaM-bound proteins whether they retain the autoinhibitory loop or
not, so the difference between, for example, CaM-bound eNOS and
CaM-bound eNOS is expected to be small. An increase in the
activities of the CaM-bound proteins caused by loop deletion is
consistent with the fact that, even in the loop-free proteins, there is
a small CaM-dependent increase in the rates of both
cytochrome c and ferricyanide reduction. If this enhancement
is caused by a conformational change that facilitates electron transfer
to the heme domain, the conformational change must occur even in the
loopless proteins to account for the CaM-dependent
incremental stimulation of activity.
P450 reductase and the NOS proteins without an autoinhibitory loop,
including the isolated iNOS reductase domain, full-length iNOS, and the
CaM-free N/I and E/I chimeras, exhibit a high electron transfer
activity as measured by their ability to reduce cytochrome c, and, in the case of the full-length NOS isoforms, to
produce ·NO (Figs. 3 and 4). Conversely, CaM-free NOS proteins
with the autoinhibitory loop possess a lower electron
transfer activity, as found for CaM-free nNOS, eNOS, and the N/E
chimera. CaM-bound proteins with the intact eNOS reductase domain also
have a relatively low electron transfer activity, although this
activity is higher than that for the CaM-free proteins. In the case of
nNOS and I/N, the electron transfer rates when CaM is bound approach
those for P450 reductase and iNOS. Likewise, the loopless eNOS and
E/I, N/I, and N/E
chimeras have enhanced reductase activities even when free of CaM. In contrast, the electron transfer activities of
eNOS, I/E, and N/E (under salt-free conditions) are low even when CaM
is bound. All of these observations are consistent with an
autoinhibitory role for the loop with respect to the binding of CaM and
the activation of electron transfer and ·NO synthesis. When the
strongly inhibitory eNOS loop is removed from these proteins, the
enzymatic activities approach, even if they do not equal, those of the
loopless iNOS isoform.
The low activity of eNOS compared with nNOS, both of which have inserts
in the FMN domain, suggests that only the eNOS autoinhibitory loop is
sufficiently effective that its impairment of electron transfer is only
partly relieved even when CaM is fully bound. The following evidence
supports this inference. 1) The electron transfer activities of
CaM-free eNOS and nNOS are lower than that of P450 reductase or the
CaM-free iNOS reductase domain expressed as an isolated polypeptide
(43). This diminution of the electron transfer activity is completely
relieved when the loop in the nNOS reductase domain is displaced by the
binding of CaM but is only partially relieved in the eNOS reductase
domain by either loop deletion or CaM binding. 2) CaM-free nNOS has a
higher intrinsic reductase activity than CaM-free eNOS, suggesting that
autoinhibition of electron transfer is less pronounced in CaM-free
nNOS. 3) The eNOS reductase activity is low even when CaM is bound,
suggesting that CaM-bound eNOS remains partially inhibited. This
residual autoinhibition is alleviated when the insert is removed, as
evidenced by the fact that the reductase activity of eNOS approaches
those of nNOS and iNOS (Fig. 5). 4) Deletion of the eNOS insert, as in
eNOS
and N/E
, curtails the CaM dependence of the reductase activity. The enhanced (or disinhibited) activity of eNOS
and N/E
, observed even in the absence of CaM, is similar to that of E/I,
N/I, and the isolated iNOS reductase domains, which have the naturally
loopless iNOS reductase domain (43).
Thus, the peptide insert has two salient effects. First, the insert
mediates a CaM-dependent inhibition that contributes to the
Ca2+ dependence of eNOS and nNOS. Second, the insert lowers
the intrinsic activity of the reductase domain. CaM-free eNOS and
N/E
therefore have higher reductase activities than even CaM-bound
eNOS. These effects may be mediated by inter- or intramolecular
mechanisms: either 1) the loop directly or through a conformational
change sterically hinders access to the reductase by cytochrome
c and ferricyanide, or 2) the loop induces a conformational
change within the reductase domain which inhibits intradomain electron
transfer; that is, removal of the loop increases intramolecular
electron transfer, perhaps in a manner similar to that of the
"hinge" domain in P450 reductase which has been suggested to
improve the efficiency of electron transfer between the flavins.
Another possibility is that the loop interferes with productive docking
of the NOS oxygenase and reductase domains and that displacement of the
loop by CaM eliminates this interference and allows proper docking. However, this possibility rationalizes the activation by CaM but does
not explain the higher intrinsic activity of the loop-free reductase in
the absence of CaM. The data, in accord with the observations of other
groups (46), indicate that the reductase activity is controlled
entirely within the reductase domain.
We provide strong evidence that the insert in the eNOS reductase domain
is an autoinhibitory structural element that affects the
Ca2+ dependence of eNOS and, by extension, also of nNOS.
The insert is largely, but not solely, responsible for the
Ca2+/CaM dependence of the constitutive NOS isoforms
because CaM is still required for ·NO synthesis by the eNOS
and N/E
chimeras. Furthermore, its absence cannot be the sole
determinant of the Ca2+ independence of iNOS because
chimeras such as I/N (25) and I/E (Fig. 3) with a loop-containing
reductase synthesize ·NO even in the presence of 5 mM EGTA, conditions under which the free Ca2+
concentration is far below the KD(app)
for Ca2+ in eNOS and nNOS. The autoinhibitory
loop also plays a critical role in governing the net electron transfer
ability of the reductase domain. In eNOS, the insert depresses
enzymatic activity by inhibiting electron transfer from the reductase
to the heme even when CaM is bound. The insert in nNOS may also act in
an autoinhibitory manner but is much weaker, in accord with the peptide
inhibition studies of Salerno et al. (26) and with the fact
that the rate of ·NO synthesis by purified nNOS, albeit high, is
still lower than that of iNOS (8, 12, 15, 16, 19, 20). This difference in the autoinhibitory potency of the eNOS and nNOS inserts may have
evolved in response to the different requirements for the synthesis of
·NO satisfied by the different isoforms in their in
vivo cellular context.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM25515.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Fax: 415-502-4728;
Email: ortiz{at}cgl.ucsf.edu.
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ABBREVIATIONS |
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The abbreviations used are:
NOS, nitric-oxide
synthase;
nNOS, neuronal nitric-oxide synthase;
eNOS, endothelial
nitric-oxide synthase;
iNOS, inducible macrophage nitric-oxide
synthase;
CaM, Ca2+-dependent calmodulin;
X/Y (N/E, I/E, E/I, and N/I) denote a protein
with the oxygenase (heme) + CaM region from NOS isoform X
and the reductase domain from NOS isoform Y, where N, E, and
I stand for nNOS, eNOS, and iNOS, respectively;
N/E and I/E
, chimeras N/E and I/E with the proposed autoinhibitory loop deleted;
P450 reductase, NADPH-cytochrome P450 reductase;
PCR, polymerase chain
reaction.
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REFERENCES |
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