The synthesis and reaction of a specific affinity label for the hydrophobic drug-binding domains of calmodulin

An affinity-labeling reagent for the two hydrophobic drug-binding domains of calmodulin has been prepared and its reaction with calmodulin characterized. The reagent, 10-(3-propionyloxysuccinimide)-2-(trifluoromethyl)phenothiazine, was shown to be very specific labeling reagent for these domains. Its specificity was demonstrated by the following observations. 1) Previous reports have shown that Ca2+ is required for phenothiazine binding to calmodulin, and here we show that the affinity-labeling reagent reacts with and inactivates calmodulin in the presence of Ca2+, but not in its absence. 2) Inclusion of trifluoperazine, fluphenazine, W-7, or 10-(3-aminopropyl)-2-(trifluoromethyl)phenothiazine in the reaction mixture protected calmodulin from inactivation by the reagent. 3) Inactivation by the reagent yielded calmodulin that was no longer retained on a phenothiazine-Sepharose column under conditions in which unreacted calmodulin was retained. 4) The measured stoichiometry of the reaction in the presence of excess reagent was 2.1 mol of reagent per mol of calmodulin which agrees well with previous reports of two high-affinity phenothiazine-binding sites on calmodulin. 5) The stoichiometry of the reaction was further confirmed by tryptic peptide maps which show two phenothiazine-labeled peptides unique to the fully reacted protein. 6) The spectral properties of the reagent, while attached to calmodulin, change in the presence of Ca2+ in a manner consistent with the known effects of Ca2+ binding by calmodulin on these hydrophobic domains. The specificity of the reagent makes it useful for further characterization of these hydrophobic binding domains on calmodulin.     

Complex of calmodulin with a ryanodine receptor target reveals a novel, flexible binding mode

Calmodulin regulates ryanodine receptor-mediated Ca(2+) release through a conserved binding site. The crystal structure of Ca(2+)-calmodulin bound to this conserved site reveals that calmodulin recognizes two hydrophobic anchor residues at a novel "1-17" spacing that brings the calmodulin lobes close together but prevents them from contacting one another. NMR residual dipolar couplings demonstrate that the detailed structure of each lobe is preserved in solution but also show that the lobes experience domain motions within the complex. FRET measurements confirm the close approach of the lobes in binding the 1-17 target and show that calmodulin binds with one lobe to a peptide lacking the second anchor. We suggest that calmodulin regulates the Ca(2+) channel by switching between the contiguous binding mode seen in our crystal structure and a state where one lobe of calmodulin contacts the conserved binding site while the other interacts with a noncontiguous site on the channel.     

The structure of the complex of calmodulin with KAR-2: a novel mode of binding explains the unique pharmacology of the drug

3'-(beta-Chloroethyl)-2',4'-dioxo-3,5'-spiro-oxazolidino-4-deacetoxyvinblastine (KAR-2) is a potent anti-microtubular agent that arrests mitosis in cancer cells without significant toxic side effects. In this study we demonstrate that in addition to targeting microtubules, KAR-2 also binds calmodulin, thereby countering the antagonistic effects of trifluoperazine. To determine the basis of both properties of KAR-2, the three-dimensional structure of its complex with Ca(2+)-calmodulin has been characterized both in solution using NMR and when crystallized using x-ray diffraction. Heterocorrelation ((1)H-(15)N heteronuclear single quantum coherence) spectra of (15)N-labeled calmodulin indicate a global conformation change (closure) of the protein upon its binding to KAR-2. The crystal structure at 2.12-A resolution reveals a more complete picture; KAR-2 binds to a novel structure created by amino acid residues of both the N- and C-terminal domains of calmodulin. Although first detected by x-ray diffraction of the crystallized ternary complex, this conformational change is consistent with its solution structure as characterized by NMR spectroscopy. It is noteworthy that a similar tertiary complex forms when calmodulin binds KAR-2 as when it binds trifluoperazine, even though the two ligands contact (for the most part) different amino acid residues. These observations explain the specificity of KAR-2 as an anti-microtubular agent; the drug interacts with a novel drug binding domain on calmodulin. Consequently, KAR-2 does not prevent calmodulin from binding most of its physiological targets.     

Model for the interaction of amphiphilic helices with troponin C and calmodulin

Crystals of troponin C are stabilized by an intermolecular interaction that involves the packing of helix A from the N-terminal domain of one molecule onto the exposed hydrophobic cleft of the C-terminal domain of a symmetry related molecule. Analysis of this molecular recognition interaction in troponin C suggests a possible mode for the binding of amphiphilic helical molecules to troponin C and to calmodulin. From the template provided by this troponin C packing, it has been possible to build a model of the contact region of mastoporan as it might be bound to the two Ca2+ binding proteins. A possible binding mode of melittin to calmodulin is also proposed. Although some of the characteristics of binding are similar for the two amphiphilic peptides, the increased length of melittin requires a significant bend in the calmodulin central helix similar to that suggested recently for the myosin light chain kinase calmodulin binding peptide (Persechini and Kretsinger: Journal of Cardiovascular Pharmacology 12:501-512, 1988). Not only are the hydrophobic interactions important in this model, but there are several favorable electrostatic interactions that are predicted as a result of the molecular modeling. The regions of troponin-C and calmodulin to which amphiphilic helices bind are similar to the regions to which the neuroleptic drugs such as trifluoperazine have been predicted to bind (Strynadka and James: Proteins 3:1-17, 1988).     

A fluorescent calmodulin that reports the binding of hydrophobic inhibitory ligands.

Ca2+ binding to calmodulin in the pCa range 5.5-7.0 exposes hydrophobic sites that bind hydrophobic inhibitory ligands, including calmodulin antagonists, some Ca2+-antagonists and calmodulin-binding proteins. The binding of these hydrophobic ligands to calmodulin can be followed by the approx. 80% fluorescence increase they produce in dansylated (5-dimethylaminonaphthalene-1-sulphonylated) calmodulin (CDRDANS). In the presence of Ca2+, calmodulin binds the calmodulin inhibitor, R24571, with an affinity of approx. 2-3 nM and hydrophobic ligands, including trifluoperazine (TFP), W-7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide], fendiline, felodipine and prenylamine, with affinities in the micromolar range. This binding is strongly Ca2+-dependent and Mg2+-independent. Calmodulin shows a reasonably high degree of specificity in its binding of these ligands over other ligands tested. CDRDANS, therefore, provides a convenient and simple means of monitoring the interaction of a variety of hydrophobic ligands with the Ca2+-dependent regulatory protein, calmodulin. CDRDANS binds to phospholipid vesicles made of (dimyristoyl)phosphatidylcholine (DMPC) or (dipalmitoyl)phosphatidylcholine (DPPC) and produces fluorescence increases only in the presence of Ca2+ and at temperatures above their gel-to-liquid crystalline phase transition. Although the fluorescence changes in CDRDANS accurately report phase transitions in these liposomes, its binding to these vesicles is weak. Calmodulin probably requires a high-affinity lipid-bound receptor protein for its high-affinity binding to natural membranes.     

Structure and dynamics of calmodulin in solution.

To characterize the dynamic behavior of calmodulin in solution, we have carried out molecular dynamics (MD) simulations of the Ca2+-loaded structure. The crystal structure of calmodulin was placed in a solvent sphere of radius 44 A, and 6 Cl- and 22 Na+ ions were included to neutralize the system and to model a 150 mM salt concentration. The total number of atoms was 32,867. During the 3-ns simulation, the structure exhibits large conformational changes on the nanosecond time scale. The central alpha-helix, which has been shown to unwind locally upon binding of calmodulin to target proteins, bends and unwinds near residue Arg74. We interpret this result as a preparative step in the more extensive structural transition observed in the "flexible linker" region 74-82 of the central helix upon complex formation. The major structural change is a reorientation of the two Ca2+-binding domains with respect to each other and a rearrangement of alpha-helices in the N-terminus domain that makes the hydrophobic target peptide binding site more accessible. This structural rearrangement brings the domains to a more favorable position for target binding, poised to achieve the orientation observed in the complex of calmodulin with myosin light-chain kinase. Analysis of solvent structure reveals an inhomogeneity in the mobility of water in the vicinity of the protein, which is attributable to the hydrophobic effect exerted by calmodulin's binding sites for target peptides.     

Two trifluoperazine-binding sites on calmodulin predicted from comparative molecular modeling with troponin-C

Among the known regulatory proteins that are conformationally sensitive to the binding of calcium ions, calmodulin and troponin-C have the greatest primary sequence homology. This observation has led to the conclusion that the most accurate predicted molecular model of calmodulin would be based on the X-ray crystallographic coordinates of the highly refined structure of turkey skeletal troponin-C. This paper describes the structure of calmodulin built from such a premise. The resulting molecular model was subjected to conjugate gradient energy minimization to remove unacceptable intramolecular non-bonded contacts. In the analysis of the resulting structure, many features of calmodulin, including the detailed conformation of the Ca2+-binding loops, the amino- and carboxy-terminal hydrophobic patches of the Ca2+-bound form, and the several clusters of acidic residues can be reconciled with much of the previously published solution data. Calmodulin is missing the N-terminal helix characteristic of troponin-C. The deletion of three residues from the central helical linker (denoted D/E in troponin-C) shortens the molecule and changes the orientation of the two domains of calmodulin by 60 degrees relative to those in troponin-C. The molecular model has been used to derive two possible binding sites for the antipsychotic drug trifluoperazine, a potent competitive inhibitor of calmodulin activity.     

Structure of calmodulin refined at 2.2 A resolution

The crystal structure of mammalian calmodulin has been refined at 2.2 A (1 A = 0.1 nm) resolution using a restrained least-squares method. The final crystallographic R-factor, based on 6685 reflections in the range 2.2 A less than or equal to d less than or equal to 5.0 A with intensities exceeding 2.5 sigma, is 0.175. Bond lengths and bond angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 1.7 degrees, respectively. The refined model includes residues 5 to 147, four Ca2+ and 69 water molecules per molecule of calmodulin. The electron density for residues 1 to 4 and 148 is poorly defined, and they are not included in the model. The molecule is shaped somewhat like a dumbbell, with an overall length of 65 A; the two lobes are connected by a seven-turn alpha-helix. Prominent secondary structural features include seven alpha-helices, four Ca2+-binding loops, and two short, double-stranded antiparallel beta-sheets between pairs of adjacent Ca2+-binding loops. The four Ca2+-binding domains in calmodulin have a typical EF hand conformation (helix-loop-helix) and are similar to those described in other Ca2+-binding proteins. The X-ray structure determination of calmodulin shows a large hydrophobic cleft in each half of the molecule. These hydrophobic regions probably represent the sites of interaction with many of the pharmacological agents known to bind to calmodulin.     

Molecular dynamics simulation of the calmodulin-trifluoperazine complex in aqueous solution

Trifluoperazine (TFP) has been widely studied in relation to its mode of binding and its inactivation of calmodulin (CaM). Most studies in solution have indicated that CaM has two high-affinity binding sites for TFP. The crystal structure of the 1:4 CaM-TFP complex (CaM-4TFP) shows that three TFP molecules bind to the C-domain of CaM, and that one TFP molecule binds to the N-domain. In contrast, the crystal structure of the 1:1 CaM-TFP complex (CaM-1TFP) shows that one TFP molecule binds to the C-domain. It has been thought that the binding of one TFP molecule to the C-domain is followed by binding to the N-domain. The crystal structure of the 1:2 CaM-TFP complex (CaM-2TFP), moreover, has recently been determined, showing that two TFP molecules bind to the C-domain. In order to determine the structure of the CaM-TFP complex and to clarify the interaction between CaM and TFP in solution, we performed a molecular dynamics simulation of the CaM-TFP complex in aqueous solution starting from the CaM-4TFP crystal structure. The obtained solution structure is very similar to the CaM-2TFP crystal structure. The computer simulation showed that the binding ability of the secondary binding site of the C-domain is higher than that of the primary binding site of the N-domain.     

A model for human cardiac troponin C and for modulation of its Ca2+ affinity by drugs

Calcium sensitizers are drugs which increase force development in striated muscle by sensitizing myofilaments to Ca2+. This can happen by increasing Ca2+ affinity of the regulatory domain of Ca2+ binding protein troponin C. High resolution crystal structures of two calcium binding proteins, calmodulin (Babu et al.: J. Mol. Biol. 203:191-204, 1988) and skeletal troponin C (Satyshur et al.: J. Biol. Chem. 263:1628-1647, 1988; Herzber et al.: J. Mol. Biol. 203:761-779, 1988), have recently been published. This makes it possible to model in detail the calcium-sensitizing action of drugs on troponin C. In this study a model of human cardiac troponin C in three-calcium state has been constructed. When calcium is bound to calcium site II of cardiac troponin C an open conformation of the protein results, which has a hydrophobic pocket surrounded by a few polar side chains. Complexation of three drugs, trifluoperazine, bepridil, and pimobendan, to the hydrophobic pocket is studied using energy minimization techniques. Two different binding modes are found, which differ in the location of a strong electrostatic interaction. In analogy with the crystal structure of skeletal troponin C it is hypothezed that in cardiac troponin C an interaction occurs between Gln-50 and Asp-88, which has a long-range effect on calcium binding. The binding modes of drugs, where a strong interaction with Asp-88 exists, can effectively prevent the interaction between Asp-88 and Gln-50 in the protein, and are proposed to be responsible for the calcium-sensitizing properties of the studied drugs.     

Calmodulin binding to alpha 1-purothionin: solution binding and modeling of the complex.

CD and fluorescence spectroscopic measurements show that calmodulin (CaM) binds to purothionins (alpha 1-purothionin: alpha 1-PT; beta-purothionin: beta-PT) in 1:1 stoichiometry with an affinity similar to that exhibited with the tightest binding CaM-binding peptides. Using the available crystal structures of CaM and alpha 1-PT, a model has been built for the interaction of CaM and alpha 1-PT and subjected to potential energy minimization. In the model, there is a bend in the central helix of CaM similar to that suggested by Persechini and Kretsinger (J. Card. Pharm. 12:501-512, 1988). alpha 1-PT fits snugly into the cavity formed by the bent CaM molecule with each of its two helices making apolar interactions with each of the two hydrophobic clefts situated at the terminal domains of CaM. The complex is further stabilized by numerous polar and electrostatic interactions on the rims of the clefts. Our model is compared with two other similar models previously reported for the CaM complexes with other helical peptides and generalizations about the mode of CaM binding to target proteins are made, which have wide relevance to the function of CaM. By analogy, a similar model is predicted for a CaM-beta-PT complex.     

Differential trace labeling of calmodulin: investigation of binding sites and conformational states by individual lysine reactivities. Effects of beta-endorphin, trifluoperazine, and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid

The Ca2+-dependent association of beta-endorphin and trifluoperazine with porcine testis calmodulin, as well as the effects of removing Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) treatment, were investigated by the procedure of differential kinetic labeling. This technique permitted determination of the relative rates of acylation of each of the epsilon-amino groups of the seven lysyl residues on calmodulin by [3H]acetic anhydride under the different conditions. In all cases, less than 0.52 mol of lysyl residue/mol of calmodulin was modified, thus ensuring that the labeling pattern reflects the microenvironments of these groups in the native protein. Lysines 75 and 94 were found to be the most reactive amino groups in Ca2+-saturated calmodulin. In the presence of Ca2+ and under conditions where beta-endorphin and calmodulin were present at a molar ratio of 2.5:1, the amino groups of lysines 75 and 148 were significantly reduced in reactivity compared to calmodulin alone. At equimolar concentrations of peptide and protein, essentially the same result was obtained except that the magnitudes of the perturbation of these two lysines were less pronounced. With trifluoperazine, at a molar ratio to calmodulin of 2.5:1, significant perturbations of lysines 75 and 148, as well as Lys 77, were also found. These results further substantiate previous observations of a commonality between phenothiazine and peptide binding sites on calmodulin. Lastly, an intriguing difference in Ca2+-mediated reactivities between lysines 75 and 77 of calmodulin is demonstrated. In the Ca2+-saturated form of the protein, both lysines are part of the long connecting helix between the two homologous halves of the protein (Babu, Y. S., Sack, J. S., Greenhough, T. G., Bugg, C. E., Means, A. R., and Cook, W. J. (1985) Nature 315, 37-40). Yet, Lys 75 increases in reactivity some 25-fold, compared to only a 2-fold change for Lys 77, in going from EGTA-treated to Ca2+-saturated calmodulin. Thus, the microenvironment of Lys 75 is markedly altered upon Ca2+ binding, and this linker region between the two globular lobes of the protein appears to be quite important in the interaction of calmodulin with inhibitory molecules and perhaps activatable enzymes.     

The nature of the trifluoperazine binding sites on calmodulin and troponin-C

We have employed 1H-nuclear magnetic resonance spectroscopy to study the interaction of the drug trifluoperazine with calmodulin and troponin-C. Distinct trifluoperazine-binding sites exist in the N- and C-terminal halves of both proteins. Each site consists of a group of hydrophobic side-chains brought into proximity by the Ca2+-dependent juxtaposition of two alpha-helical segments of the protein, each, in turn, belonging to a different Ca2+-binding site in the protein half. The trifluoperazine-induced inhibition of the biological activating ability of calmodulin appears to result from conformational restrictions conferred upon the protein by the bound drug.     

Localization of a felodipine (dihydropyridine) binding site on calmodulin

The fluorescent dihydropyridine calcium antagonist drug felodipine binds to calmodulin (CaM) in a Ca2+-dependent manner. Its binding can be regulated by the interaction of CaM antagonist drugs through allosteric mechanisms [Mills, J.S., & Johnson, J.D. (1985) Biochemistry 24, 4897]. Here, we have examined the binding of a nonspecific hydrophobic fluorescent probe molecule TNS (toluidinylnaphthalenesulfonate) and of felodipine to CAM and several of its proteolytic fragments. While TNS interacts with sites on both the amino-terminal half of the protein [proteolytic fragment TR1C (1-77)] and carboxy-terminal half [proteolytic fragment TR2C (78-148)], felodipine binding shows more selectivity. It binds in a Ca2+-dependent manner to the proteolytic fragments TM1 (1-106) and TR2E (1-90) but exhibits only weak affinity for TR1C (1-77) and TR2C (78-148). Furthermore, felodipine exhibits selectivity over TNS and trifluoperazine (TFP) in blocking the tryptic cleavage between residues 77 and 78. These studies indicate a selective binding of felodipine to a hydrophobic site existing in residues 1-90 and suggest that productive binding requires amino acids in the region 78-90. Although the felodipine binding site is preserved in fragment 1-106, the allosteric interactions between the prenylamine and the felodipine binding sites that are observed with intact CaM are not observed in this fragment. Rather, prenylamine simply displaces felodipine from its binding site on this fragment. Our results are consistent with calmodulin containing not less than two allosterically related hydrophobic drug binding sites. One of these sites (felodipine) appears to be localized in region 1-90 and the other one in region 78-148.     

Calmodulin-peptide interactions: apocalmodulin binding to the myosin light chain kinase target-site

Noncovalent binding of the synthetic peptide RS20 to calmodulin in the presence of calcium was confirmed by electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry to form a complex with a 1:1:4 calmodulin/RS20/calcium stoichiometry. There was no evidence for formation of a calmodulin-RS20-Ca(2) species. The absence of calmodulin-RS20-Ca(2) would be consistent with models in which the two globular domains are coupled functionally. There was evidence that calmodulin, RS20-calmodulin without associated calcium, and calmodulin-RS20-Ca(4) existed together in solution, whereas calmodulin-calcium complexes were absent. It is proposed that calcium binding to form the calmodulin-RS20-Ca(4) complex occurs after an initial RS20-calmodulin binding event, and serves to secure the target within the calmodulin structure. The binding of more than one RS20 molecule to calmodulin was observed to induce unfolding of calmodulin.     

A Common Structural Basis for pH- and Calmodulin-mediated Regulation in Plant Glutamate Decarboxylase

Glutamate decarboxylase (Gad) catalyzes glutamate to γ-aminobutyrate conversion. Plant Gad is a ∼340 kDa hexamer, involved in development and stress response, and regulated by pH and binding of Ca²⁺/calmodulin (CaM) to the C-terminal domain. We determined the crystal structure of Arabidopsis thaliana Gad1 in its CaM-free state, obtained a low-resolution structure of the calmodulin-activated Gad complex by small-angle X-ray scattering and identified the crucial residues, in the C-terminal domain, for regulation by pH and CaM binding. CaM activates Gad1 in a unique way by relieving two C-terminal autoinhibition domains of adjacent active sites, forming a 393 kDa Gad1-CaM complex with an unusual 1:3 stoichiometry. The complex is loosely packed: thanks to the flexible linkers connecting the enzyme core with the six C-terminal regulatory domains, the CaM molecules retain considerable positional and orientational freedom with respect to Gad1. The complex thus represents a prototype for a novel CaM-target interaction mode. Thanks to its two levels of regulation, both targeting the C-terminal domain, Gad can respond flexibly to different kinds of cellular stress occurring at different pH values.     

First structural evidence of a specific inhibition of phospholipase A2 by alpha-tocopherol (vitamin E) and its implications in inflammation: crystal structure of the complex formed between phospholipase A2 and alpha-tocopherol at 1.8 A resolution

This is the first structural evidence of alpha-tocopherol (alpha-TP) as a possible candidate against inflammation, as it inhibits phospholipase A2 specifically and effectively. The crystal structure of the complex formed between Vipera russelli phospholipase A2 and alpha-tocopherol has been determined and refined to a resolution of 1.8 A. The structure contains two molecules, A and B, of phospholipase A2 in the asymmetric unit, together with one alpha-tocopherol molecule, which is bound specifically to one of them. The phospholipase A2 molecules interact extensively with each other in the crystalline state. The two molecules were found in a stable association in the solution state as well, thus indicating their inherent tendency to remain together as a structural unit, leading to significant functional implications. In the crystal structure, the most important difference between the conformations of two molecules as a result of their association pertains to the orientation of Trp31. It may be noted that Trp31 is located at the mouth of the hydrophobic channel that forms the binding domain of the enzyme. The values of torsion angles (phi, psi, chi(1) and chi(2)) for both the backbone as well as for the side-chain of Trp31 in molecules A and B are -94 degrees, -30 degrees, -66 degrees, 116 degrees and -128 degrees, 170 degrees, -63 degrees, -81 degrees, respectively. The conformation of Trp31 in molecule A is suitable for binding, while that in B hinders the passage of the ligand to the binding site. Consequently, alpha-tocopherol is able to bind to molecule A only, while the binding site of molecule B contains three water molecules. In the complex, the aromatic moiety of alpha-tocopherol is placed in the large space at the active site of the enzyme, while the long hydrophobic channel in the enzyme is filled by hydrocarbon chain of alpha-tocopherol. The critical interactions between the enzyme and alpha-tocopherol are generated between the hydroxyl group of the six-membered ring of alpha-tocopherol and His48 N(delta1) and Asp49 O(delta1) as characteristic hydrogen bonds. The remaining part of alpha-tocopherol interacts extensively with the residues of the hydrophobic channel of the enzyme, giving rise to a number of hydrophobic interactions, resulting in the formation of a stable complex.     

Fluorescence investigations of calmodulin hydrophobic sites

Calmodulin activation of target enzymes depends on the interaction between calmodulin hydrophobic regions and some enzyme areas. The Ca2+ induced exposure of calmodulin hydrophobic sites was studied by means of 2-p-toluidinylnaphthalene-6-sulfonate, a fluorescent probe. Scatchard and Job plots showed that the calmodulin-Ca42+ complex bound two molecules of this hydrophobic probe, with KD congruent to 1.4 X 10(-4) M. These sites are not totally exposed until calmodulin has bound four Ca2+ per molecule, so the conformational change is not over before the four specific Ca2+ - binding sites are saturated with Ca2+.     

Protein binding properties of some histamine H2 antagonists: a 1H nuclear magnetic resonance study of antihistamine-calmodulin interactions

Oxmetidine (SK & F 92994) is a potent histamine H2 antagonist, which, however, also demonstrates cardiac effects consistent with its inhibiting transmembrane calcium fluxes. 1H nuclear magnetic resonance has been used to show that oxmetidine binds to a single site on the regulatory calcium-binding protein, calmodulin. Binding requires the presence of at least two equivalents of calcium per mol protein, is characterized by fast exchange behaviour and a dissociation constant of about 4 mM and is not affected by the presence of trifluoperazine. Protein-induced spectral changes and a limited study of structure-affinity relationships suggest the importance of the drug imidazole and benzyldioxymethylene groups in determining the strength of the interaction. Drug-induced perturbations in the spectrum of calmodulin indicate that the binding site is in the C-terminal half of the protein, and involves a hydrophobic area containing His-107, Met-144, Met-145 and possibly Phe-89, Phe-141, and calcium binding site III.