©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Calmodulin-Nitric Oxide Synthase Interaction
CRITICAL ROLE OF THE CALMODULIN LATCH DOMAIN IN ENZYME ACTIVATION (*)

(Received for publication, May 18, 1995; and in revised form, August 8, 1995)

Zenghua Su Michael A. Blazing (§) Daju Fan Samuel E. George (¶)

From the Departments of Medicine and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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^G-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.


INTRODUCTION

Nitric oxide synthases (NOS) (^1)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.


EXPERIMENTAL PROCEDURES

Construction of Mutant CaM Constructs

We used standard mutagenesis techniques to construct the CaM-cTnC chimeras and CaM mutants(15, 16, 19) .

Purification of NOS and Assay of NOS Activity

Nitric oxide synthase was purified from porcine brain(4, 20) . We also used 293 cells stably transfected with rat brain nNOS cDNA (gift of Solomon Snyder) as a source of enzyme. Enzyme preparations were >90% pure as judged by Coomassie Blue staining and free of calmodulin as judged by immunoblots. NOS activity was determined by conversion of [^3H]arginine to [^3H]citrulline, as described by Bredt and Snyder(21) .

Evaluation of Chimeras as Inhibitors

NOS activity was determined at five different CaM concentrations: 9, 15, 22.5, 30, and 60 nM. Each of the five CaM concentrations was then evaluated with 4-6 different concentrations of the putative inhibitor, ranging from 30 to 1200 nM. The precise concentrations of inhibitory chimera chosen varied depending on the strength of the observed competitive inhibition. Each point was determined in duplicate. After subtracting background ([^3H]citrulline present in a sample with Ca-CaM omitted and containing 1 mM EGTA), the data were recorded as a percentage of maximal activation (defined as activation observed with 60 nM CaM and no inhibitor) and plotted on double-reciprocal plots. Data points were fit to a line using least squares analysis (Marquardt algorithm, Kaleidagraph, Synergy Software, Reading, PA); all regression coefficients were 0.97 or higher. K(i) was calculated by the formula K(i) = [I]/((K/K) - 1), where [I] is the concentration of inhibitory chimera, 1/K is the calculated x axis intercept in the presence of inhibitor, and 1/K is the calculated x axis intercept when no inhibitor was present. All data points were obtained in duplicate and the experiments were repeated at least twice. Each concentration of inhibitor tested yielded a K(i) value; the mean of these K(i) values ± S.E. was determined.

Additive Inhibition of nNOS by CaM-cTnC Chimeras and NMMA/7-Nitroindazole

N^G-Monomethyl-L-arginine (NMMA) was obtained from Calbiochem and 7-nitroindazole from Tocris Cookson (Langford, UK). Activity of nNOS was determined with the CaM and arginine concentrations held constant at 60 and 120 nM, respectively. NMMA or 7-nitroindazole was added to the samples in progressively larger concentrations, as indicated. The assay was then repeated in the presence of 60 and 105 nM CaM (1 TnC)(BM1). Maximal activation is defined as the amount of [^3H]citrulline present in samples with 60 nM CaM and no inhibitors present, after background counts were subtracted.

Displacement of I-CaM from Immobilized nNOS

Purified nNOS (10 ng/lane) was run on a 7.5% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane by electrophoresis. A small amount of prestained high molecular weight standards (Bio-Rad) was added so that the precise lateral extent of each lane could be visually identified. After transfer, the nitrocellulose membrane was soaked for 2 h at room temperature in 5% nonfat dry milk in 50 mM Tris-Cl, pH 7.5, 0.2 M NaCl, 0.5 mM CaCl(2), 50 mM MgCl(2), 0.05% Tween 20 (buffer A). The membrane was cut into strips using the prestained markers as a guide and then incubated with shaking for 6 h at room temperature with I-CaM (obtained from DuPont NEN, labeled by Bolton-Hunter method; 4 times 10^6 cpm) in 2 ml of buffer A, along with increasing competitive amounts of CaM or CaM mutant as indicated. Each sample was done in triplicate. The strips were washed 5 times for 15 min each in buffer A, and then the amount of bound I-CaM was determined by counting. After subtracting background (I-CaM bound to a strip with prestained markers only), the percentage of counts present on a strip relative to the counts present on the strip incubated without competitor (i.e.I-CaM alone) was determined.


RESULTS

Activation of nNOS by CaM-cTnC Chimeras

cTnC showed no ability to activate nNOS at concentrations up to 5 µM (data not shown). To localize the regions of cTnC that were responsible for its inability to activate nNOS, we constructed a series of CaM-cTnC chimeras (Fig. 1) and assayed these chimeric proteins for their ability to activate nNOS (Table 1).


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.

Competitive Inhibition of nNOS by CaM-cTnC Chimeras

Two explanations could account for a failure to activate. First, the chimera may simply fail to bind the enzyme. Alternatively, it may bind but fail to induce conformational changes necessary for activation. A CaM mutant that cannot bind should neither activate nNOS nor compete with CaM for nNOS binding. A mutant that binds to nNOS but cannot activate should competitively inhibit nNOS activation by CaM.

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(i) 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(i) of about 120 nM. Restoration of the inactive first EF-hand of CaM (1 TnC) reduced its K(i) 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(i) than TnC (3,4 CaM) (44 and 198 nM, respectively). Similarly, CaM (1 TnC)(BM1) has a 4.5-fold lower K(i) 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.



NOS-activating Residues Cluster in the Latch Domain of Calmodulin

Our data show that domains 1 and 3 of CaM contain amino acid residues that are essential for nNOS activation and cannot be substituted by the corresponding residues of cTnC. To establish the identity of these residues, we performed additional mutagenesis on CaM domains 1 and 3, substituting helix-loop-helix subdomains and individual cTnC residues for the corresponding subdomains and residues of CaM.

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.


DISCUSSION

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.

Significance of Competitive Inhibition

As a consequence of the dissociation between binding and activation, our work has led to the development of a new class of nNOS inhibitors. These inhibitors are not only interesting tools for understanding the mechanism of nNOS activation but also represent a potentially useful addition to the arsenal of NOS inhibitors. Since they work through a mechanism that is distinct from existing inhibitors, they are additive with existing inhibitors and can therefore promote more complete inhibition. In addition, this class of inhibitors can be delivered genetically, possibly in a targeted way.

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(i) 118 nM) with TnC (3,4 CaM) (K(i) 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(i) 26 nM) with TnC (3,4 CaM)(BM1) (K(i) 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.

Differential Effects of CaM Mutations on Target Enzymes, Implications for nNOS Activation

Are the observed mutational effects specific for nNOS or do they reflect a general mechanism through which CaM activates its diverse target enzymes? We have evaluated this panel of CaM-cTnC chimeras and related mutants on five CaM target enzymes (smooth muscle myosin light chain kinase, calcium/calmodulin-regulated phosphodiesterase, calcium/calmodulin-dependent multifunctional protein kinase (CaM kinase II), and calcineurin(15, 16, 19) . (^2)We have consistently observed striking differential effects on CaM target enzymes (Table 4). For example, although CaM (1 TnC) and CaM (1 TnC)(BM 1) are potent nNOS antagonists, they substitute perfectly for CaM in the activation of PDE and act as partial agonists toward CaM kinase II(15) .



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.

A Hypothesis for Latch Domain-nNOS Interactions

The differential effects of CaM mutations stand in marked contrast to the crystal structure of CaM complexed with various peptides modeled after the CaM-binding domains of target enzymes. The CaM-peptide structures show a great deal of similarity, despite considerable divergence in the sequences of the peptides(18, 23, 24) . Moreover, the affinity of CaM for model peptides is fairly constant, despite their diverse origins (typical K(d) values of about 0.5-3 nM(26, 27, 28, 29, 30, 31) ). These observations suggest that CaM binding to model peptides follows a general mechanism, driven primarily by strong hydrophobic interactions that are relatively independent of the peptide's exact primary sequence(18, 23) .

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.




FOOTNOTES

*
This work was supported by United States Public Health Service Grant HL48662 and a grant-in-aid from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Clinician Scientist of the American Heart Association.

Established Investigator of the American Heart Association. To whom correspondence should be addressed: Cardiovascular Division, Box 3060, Duke University Medical Center, Durham, NC 27710. Tel.: 919-681-8446; Fax: 919-684-8591. seg@hodgkin.mc.duke.edu.

(^1)
The abbreviations used are: NOS, nitric oxide synthase; nNOS, rat neuronal isoform of nitric oxide synthase; CaM, calmodulin; TnC, troponin C; BM1, binding mutant domain 1; Parv, parvalbumin; cTnC, cardiac troponin C; PDE, calmodulin-stimulated cyclic nucleotide phosphodiesterase; smMLCK, smooth muscle myosin light chain kinase; CaM kinase II, multifunctional calmodulin-dependent protein kinase; NMMA, N^G-monomethyl-L-arginine.

(^2)
S. George and Z. Su, unpublished observations.


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