(Received for publication, May 18, 1995; and in revised form, August 8, 1995)
From the
The neuronal isoform of nitric oxide synthase (nNOS) requires
calmodulin for nitric oxide producing activity. Calmodulin functions as
a molecular switch, allowing electron transport from the
carboxyl-terminal reductase domain of nitric oxide synthase to its
heme-containing amino-terminal domain. Available evidence suggests that
calmodulin binds to a site between the two domains of nNOS, but it is
not known how calmodulin then executes its switch function. To study
the calmodulin-nNOS interaction, we created a series of chimeras
between calmodulin and cardiac troponin C (cTnC, a homologue of
calmodulin that does not activate nNOS). Although a few chimeras showed
good ability to activate nNOS, most failed to activate. A subset of the
inactive chimeras retained the ability to bind to nNOS and therefore
functioned as potent competitive inhibitors of nNOS activation by
calmodulin (CaM). The observed inhibition was additive with the
arginine antagonists N-monomethyl-L-arginine and
7-nitroindazole, indicating a distinct and independent mechanism of
nNOS inhibition. To localize the calmodulin residues that account for
impaired activation in the inhibitory CaM-cTnC chimeras, we conducted a
detailed mutagenesis study, replacing CaM subdomains and individual
amino acid residues with the corresponding residues from cTnC. This
revealed that mutations in CaM helices 2 and 6 (its latch domain) have
a disproportionate negative effect on nNOS activation. Thus, our
evidence suggests that the CaM latch domain plays a critical role in
its molecular switch function.
Nitric oxide synthases (NOS) ()are reversibly
regulated by calcium calmodulin (CaM) or alternatively possess a
tightly bound CaM subunit (reviewed in (1, 2, 3) ). The molecular mechanism through
which CaM binds and activates NOS is currently a subject of active
investigation. One current model suggests a bidomain structure for NOS,
with a carboxyl-terminal domain homologous to cytochrome P450
reductases and a heme-containing amino-terminal domain. A consensus
CaM-binding site is situated more or less in the center of NOS and
separates the two domains(4) . There is substantial
experimental evidence that calmodulin is required for electron flow,
from NADPH into the reductase domain (5) and from the reductase
domain to the heme
domain(5, 6, 7, 8) . This has led to
the proposal that CaM functions as a molecular switch, inducing a
conformational change in NOS that facilitates electron
flow(7) . How CaM fulfills this switch function is currently
unknown.
Calmodulin is a member of a superfamily of structurally related calcium signaling proteins (reviewed in (9, 10, 11) ). Members of this superfamily share a 29-residue helix-loop-helix motif called the EF-hand that is responsible for high affinity calcium binding(9, 10) . Calmodulin has four EF-hand domains, arranged as two pairs separated by an eight-turn central helix(9) . A closely related calcium signaling protein, troponin C, is 50% identical to calmodulin and has a strikingly similar crystal structure(12, 13, 14) . Nevertheless, CaM and troponin C substitute poorly for each other as signaling molecules. Troponin C generally cannot activate calmodulin target enzymes(15, 16) , and CaM does not substitute well in reconstituted thin filament systems(17) . Structural similarity but functional divergence makes the construction of chimeras a useful approach for structure-function studies.
In this study, we evaluate the interaction of CaM, the cardiac isoform of TnC (cTnC), and a series of CaM-cTnC chimeras with the rat brain isoform of NOS (nNOS)(4) . We find that although cTnC neither binds nor activates nNOS, most CaM-cTnC chimeras at least retain the ability to bind to nNOS; moreover, some are reasonably good nNOS activators as well. Notably, we find an interesting subset of the CaM-cTnC chimeras that bind to nNOS but fail to activate and therefore function as potent competitive inhibitors of nNOS activation by CaM. Finally we extend our mutagenesis strategy to show that mutations in the helix 2-helix 6 region of CaM, its so-called latch domain(18) , seriously impair activation of nNOS. Our data demonstrate that the latch domain of CaM is an important interaction site between CaM and nNOS and plays a critical role in nNOS activation.
Figure 1:
Sequence alignment of CaM and cTnC and
examples of CaM-cTnC chimeras. Top, the amino acid sequences
of CaM and the cardiac isoform of cTnC are aligned according to
similarity in primary and secondary structure. Locations of the four
EF-hand domains and subdomain loops and helices are as indicated (here,
the helices are numbered 1-8; they are also commonly
named helices A-H. Arrows indicate splice points for
constructing chimeras. Residue numbers are indicated below the sequence. The central helices of CaM and cTnC, a fusion of
helices 4 and 5, are indicated. Bottom, examples of some of
the CaM-cTnC chimeras used in this study. Sequences derived from CaM
are white; those from cTnC are shaded. An x inside loop 1 indicates that the loop is nonfunctional, having its
origins as the nonfunctional first loop of the cardiac isoform of cTnC.
The designation BM1 (Binding Mutant domain 1) indicates that
Ca binding in the nonfunctional first loop has been
restored by mutagenesis.
None of the half-CaM, half-cTnC chimeras showed any ability to activate nNOS. Thus, substitution of either half of CaM with the corresponding domain of cTnC leads to loss of activation ability. We also substituted domains 3 and 4 of CaM with the corresponding region from rat parvalbumin, another member of the calmodulin superfamily. This chimera (CaM (3,4 Parv)) also failed to activate nNOS.
The first EF-hand of
cTnC cannot bind Ca(15, 22) ; we
thought that restoration of the loop might restore some ability to
activate. However, the mutated chimera TnC (3,4 CaM)(BM1) also failed
to activate nNOS. Thus, a functional first calcium-binding domain is
not sufficient to confer nNOS activation.
We next evaluated a series of CaM-cTnC chimeras in which only a single EF-hand domain was exchanged. All chimeras with only a single domain obtained from CaM (i.e. TnC (1 CaM) through TnC (4 CaM)) failed to activate. However, chimeras that contained only a single domain from cTnC showed variable ability to activate nNOS. CaM (2 TnC) and CaM (CH TnC) showed very well preserved activation, reaching over 85% of the maximal activation observed with CaM. CaM (4 TnC) was a partial nNOS agonist, showing 48% of maximal activation. In contrast, CaM (1 TnC) and CaM (3 TnC) failed to activate. CaM (1 TnC)'s impaired activation phenotype was not due to its nonfunctional first loop since its restoration (to yield CaM (1 TnC)(BM1)) did not restore activation.
The results shown in Table 1indicate that whereas three single domain CaM-cTnC chimeras retained moderate to good nNOS activation, the majority of these constructs have virtually no ability to activate. These data also point to a particularly important role for CaM domains 1 and 3, since substitution of either CaM domain 1 or CaM domain 3 with the corresponding cTnC domain was sufficient to abolish activation.
To distinguish between these two possibilities,
we determined whether the nonactivating chimeras could inhibit
CaM-stimulated nNOS activity. These results are shown in Fig. 2.
cTnC, TnC (1 CaM) through TnC (4 CaM), and CaM (3,4 Parv) did not
inhibit. In contrast, all of the half-CaM, half-cTnC chimeras were
strong inhibitors of CaM-induced NOS activation (K values from 44 to 198 nM).
Figure 2:
CaM-cTnC chimeras competitively inhibit
nNOS activation by CaM. Left, double-reciprocal plot of NOS
activation by CaM, alone and in the presence of increasing amounts of
CaM (1 TnC)(BM1). Lines were fit by least squares analysis as
described under ``Experimental Procedures.'' Right,
table showing inhibition data for cTnC and all chimeras. No
inhibition indicates that the presence of up to 1200 nM of the indicated protein did not detectably reduce the nNOS
activation observed with 60 nM CaM. Data were analyzed and K values determined as described under
``Experimental Procedures.''
The single domain chimeras
CaM (1 TnC) and CaM (3 TnC) were also strong competitive inhibitors,
each with a K of about 120 nM.
Restoration of the inactive first EF-hand of CaM (1 TnC) reduced its K
to 26 nM, making CaM (1 TnC)(BM1) the
strongest of this class of competitive nNOS inhibitors.
Restoration
of the inactive first calcium-binding loop substantially enhances
competitive inhibition. This is illustrated by the following
observations. TnC (3,4 CaM)(BM1) has a 4.5-fold lower K than TnC (3,4 CaM) (44 and 198 nM, respectively).
Similarly, CaM (1 TnC)(BM1) has a 4.5-fold lower K
than CaM (1 TnC) (26 and 118 nM, respectively). Thus,
restoration of the first calcium-binding loop enhances the ability to
interact with nNOS by 4.5-fold.
We also determined the ability of CaM (1 TnC)(BM1) to add to nNOS inhibition produced by NMMA or 7-nitroindazole under assay conditions in which calmodulin and arginine concentrations were held constant (Fig. 3). For example, 1.2 µM NMMA reduced nNOS activation to 50% of maximal; 1.2 µM NMMA plus 105 nM CaM (1 TnC)(BM1) reduced activation to <25% of maximal. This relationship was fairly constant; CaM (1 TnC)(BM1) at 105 nM more or less doubled the amount of inhibition observed at a given level of NMMA or 7-nitroindazole. Their additive nature indicates that the molecular mechanisms of inhibition are distinct and independent.
Figure 3: Inhibition produced by CaM-cTnC chimeras is additive with NMMA and 7-nitroindazole. Enzyme activity was determined as described under ``Experimental Procedures.'' CaM concentration was held constant at 60 nM and arginine concentration at 120 nM for all data points. Maximal activation is defined as that observed with 60 nM CaM in the absence of NMMA, 7-nitroindazole, or CaM (1 TnC)(BM1). Addition of increasing amounts of NMMA or 7-nitroindazole results in progressive reduction of nNOS activity; addition of CaM (1 TnC)(BM1) results in parallel downward shifts in nNOS activity curves.
We also
evaluated interactions between some of the CaM-cTnC chimeras and nNOS
by determining the ability of chimeras to displace I-labeled calmodulin from nNOS immobilized on
nitrocellulose (Table 2). Both CaM (1 TnC) and CaM (1 TnC)(BM1)
effectively competed with labeled CaM for binding to immobilized nNOS;
in contrast, TnC (1 CaM) and cTnC failed to compete.
Domain 1 of CaM and cTnC is composed of helix 1, loop 1, and helix 2. Amino acid differences in any one of these subdomains may account for the loss of nNOS activation. The differences in loop 1 do not appear to account for the inactive phenotype since restoration of a functional first loop does not restore activation. Although three amino acid differences between CaM and cTnC remain after loop restoration (K21A, G23E, and T26C), these seem unlikely to be involved in direct activating interactions with nNOS since these residues are extensively involved in loop-loop interactions with the neighboring member of the EF-hand pair(9) . cTnC has eight additional residues at its amino terminus, but removal of these additional residues from CaM (1 TnC) (15) does not restore nNOS activation (data not shown).
Helix 1 point mutations had little or no effect on nNOS activation (Table 3), but substitutions in helix 2 of calmodulin were less well tolerated. The mutations T34K and S38M both reduced activation to 70% of maximal, while the double mutant T34K/S38M reduced activation to 38% of maximal. The mutation A46E, which lies in the helix 2-helix 3 linker region, also had a moderate negative impact on activation (72% of maximal). Thus, our data show that the inability of CaM (1 TnC) to activate nNOS results primarily from three amino acid substitutions, i.e. T34K, S38M, and A46E.
We conducted a similar
mutagenesis study of domain 3. Two subdomain chimeras, CaM (helix 5
TnC) and CaM (loop 3 TnC), retained good ability to activate nNOS. In
contrast, CaM (helix 6 TnC) was significantly impaired, reaching only
32% of maximal activation. We then mutated CaM residues 107-112
to the corresponding cTnC residue. Two mutations, H107I and K115T, had
no detrimental impact on activation. Two mutations, M109L and T110Q,
had modest detrimental impact (90 and 88% of maximal activation). Two
mutations, V108M and N111A, had somewhat larger detrimental effects
(each mutant reaching 80% of maximal activation). The most striking
effect was observed with the mutation L112T, which reduced activation
to 36% of maximal and produced a significant rightward shift in K. Thus, our data show that the inability of CaM
(3 TnC) to activate nNOS is attributable largely to the substitution of
helix 6 and especially to residue Leu-112.
To confirm that residue Leu-112 had a critical role in nNOS activation, we made two back mutations. The first, CaM (3 TnC)(T112L), is identical to CaM (3 TnC), except that residue Thr-112 has been mutated to Leu. This single back mutation was sufficient to convert CaM (3 TnC) from an antagonist to a partial agonist (32% of maximal activation). The second, CaM (helix 6 TnC)(T112L), is identical to CaM (helix 6 TnC) except that residue Thr-112 has been back mutated to Leu. Again, restoration of residue Leu-112 substantially improved activation, from 32 to 82% of maximal.
The three-dimensional structures of calmodulin complexed to various model CaM-binding peptides have been determined(18, 23, 24) . These structures show that CaM envelops the model peptide in a hydrophobic tunnel by bending and twisting sharply about its central helix. The top of this tunnel, referred to as the latch domain(18, 19) , is formed by a tight junction between helixes 2 and 6 of calmodulin. Six individual CaM to cTnC mutations impair nNOS activation by 20% or more: T34K (70% of maximal), S38M (67%), A46E (72%), V108M (80%), N111A (80%), and L112T (36%). All of the residues cluster in or near the latch domain. Of these residues, only two (Val-108 and Leu-112) are interior hydrophobic residues that interact directly with CaM-binding peptides(18, 23) . The remainder are solvent-exposed residues whose side chains orient away from the peptide(18) . Therefore, they have the potential to interact with nNOS at sites other than its canonical CaM-binding domain.
CaM binding has been shown to be necessary for electron transfer from the reductase domain to the heme group of nNOS(6) . One current model suggests that CaM functions as a molecular switch, promoting electron flow from NADPH through the flavins to the heme(6, 8, 25) . Although CaM binding induces a significant conformational change in nNOS(7, 8) , the molecular mechanism through which CaM executes the switching function remains to be established.
Although the site of CaM binding to nNOS has not been directly demonstrated, a body of indirect evidence suggests that the putative CaM-binding domain of nNOS (approximately residues 720-750(4) ) is in fact a major site of interaction between CaM and nNOS(8, 26) . Since some of our mutants bind to nNOS but fail to activate, we conclude that binding alone is insufficient to promote electron transfer. Additional CaM-nNOS interactions are required for the molecular switch function of CaM. At least some of these involve the CaM latch domain.
A functional first binding loop in an inhibitory mutant enhances inhibition by a remarkably constant 4.5-fold when compared with the same mutant with nonfunctional loop. Thus, loop restoration does not restore activation but does enhance binding affinity. A possible explanation for this effect may be found in the reported CaM-peptide structures(18, 23, 24) . Although no first loop residue participates directly in binding, hydrophobic residues to the immediate amino- and carboxyl-terminal side of the first loop (i.e. Leu-18, Phe-19, and Leu-32) play a critical role in forming the hydrophobic tunnel that envelops the model peptide. The nonfunctional loop may distort these interactions and weaken binding.
The second EF-hand domain also affects the degree of
competitive inhibition. CaM (1 TnC) differs from TnC (3,4 CaM) only in
the derivation of the second domain (see Fig. 1). A comparison
of CaM (1 TnC) (K 118 nM) with TnC (3,4
CaM) (K
198 nM) reveals that the presence
of CaM's second domain enhances inhibition by 68% (198/118). The
same relationship exists with respect to the second domain because,
similarly, the presence of CaM's second domain enhances
inhibition by 69% (compare CaM (1 TnC)(BM1) (K
26
nM) with TnC (3,4 CaM)(BM1) (K
44
nM)). Thus, the presence of cTnC's second domain reduces
the efficiency of binding to nNOS. In CaM-peptide structures, a number
of CaM second domain residues participate in peptide
binding(18, 23) , and substitution of the
corresponding residue from cTnC may reduce the affinity of this
interaction. The longer central helix of cTnC may also contribute to
this effect.
Of the enzymes tested in this system, smMLCK is the most similar to nNOS in its pattern of activation(15, 16, 19) . It is the only other enzyme in which we have observed competitive inhibition, and most of the nNOS inhibitors we described here also inhibit smMLCK. However, even smMLCK and nNOS are clearly distinguishable. For example, CaM (3 TnC) is the most potent smMLCK inhibitor, whereas CaM (1 TnC)(BM1) is the most potent nNOS inhibitor. In addition, CaM (1 TnC) inhibits nNOS but acts as a partial agonist toward MLCK. Thus, at least one of the CaM-cTnC chimeras is specific for nNOS among the enzymes evaluated.
With higher resolution mutagenesis, clear differences emerge between smMLCK and nNOS. The mutations T110Q and K115T have marked effects on MLCK activation but have little or no effect on nNOS; A46E, V108M, and N111A produce moderate nNOS impairment but have no detectable effect on smMLCK. The mutations T34K, S38M, and L112T affect both enzymes, but differential effects are detectable; T34K and S38M markedly affect smMLCK but have only moderate effects on nNOS; L112T profoundly impairs nNOS but has a more moderate effect on smMLCK. Thus, although smMLCK and nNOS activation both require latch domain residues, they differ significantly in the specific residues that are involved and the degree to which they are involved. Additional mutagenesis should allow further definition of activation requirements for these enzymes and should allow the development of increasingly specific inhibitors for each enzyme.
The CaM rearrangement induced by peptide binding generates a new surface arrangement with the potential for additional target enzyme interactions. As suggested by VanBerkum and Means (32) and Kretsinger (33) , these additional interactions may be critical for enzyme activation. The data presented here and elsewhere (16, 19) demonstrate that the latch domain is a major enzyme interaction site. Moreover, the precise nature of the latch-enzyme contacts may be relatively specific from enzyme to enzyme.
A hypothesis for CaM binding and activation of nNOS is shown in Fig. 4. CaM binding to nNOS brings CaM helices 2 and 6 into close approximation and forms the latch domain. Surface residues of the latch domain then interact with residues of nNOS in the heme domain and/or reductase domain and promote a conformational change in nNOS that allows productive electron transfer. Our data establish two essential elements of the hypothesis: 1) binding and activation are separable events; and 2) mutations in the CaM latch are detrimental to nNOS activation. However, the validity of the proposed mechanism remains to be proven by additional mutagenesis and detailed structural studies.
Figure 4: A hypothesis for latch domain-nNOS interactions based upon available CaM-peptide structures and the mutagenesis and inhibition data presented in this paper. CaM binds to the canonical CaM-binding domain of nNOS. The surface of the latch domain then interacts with as yet unknown domains of nNOS, inducing nNOS into its active conformation and allowing interdomain electron transport and nitric oxide production. Inhibitory CaM mutants bind to the canonical CaM-binding domain of nNOS in a more or less normal way. However, because of latch domain mutations, they cannot induce nNOS into its active conformation.