Interacting partners of M-PMV nucleocapsid-dUTPase.

The nucleocapsid-dUTPase protein of Mason-Pfizer monkey virus is a truly bifunctional fusion enzyme. The exact role of this fusion protein in the viral life cycle is unclear. To explore its function, we started to identify interacting protein partners of the enzyme in vitro. Three viral proteins, integrase, capsid and nucleocapsid, were found to be capable of physical interaction with NC-dUTPase. Integrase protein is an important component within the preintegration complex; therefore the present results also suggest that NC-dUTPase might be associated with this complex.

Modulation of the interaction between aldolase and glycerol-phosphate dehydrogenase by fructose phosphates.

Kinetics of fructose-1,6-disphosphate aldolase (EC 4.1.2.13) catalyzed conversion of fructose phosphates was analyzed by coupling the aldolase reactions to the metabolically sequential enzyme, glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), which interacts with aldolase. At low enzyme concentration poly(ethylene glycol) was added to promote complex formation of aldolase and glycerol-phosphate dehydrogenase resulting in a 3-fold increase in KM of fructose-1,6-bisphosphate and no change in Vmax. Kinetic parameters for fructose-1-phosphate conversion changed inversely upon complex formation: Vmax increased while KM remained unchanged. Gel penetration and ion-exchange chromatographic experiments showed positive modulation of the interaction of aldolase and dehydrogenase by fructose-1,6-bisphosphate. The dissociation constant of the heterologous enzyme complex decreased 10-fold in the presence of this substrate. Fructose-1-phosphate or dihydroxyacetone phosphate had no effect on the dissociation constant of the aldolase-dehydrogenase complex. In addition, titration of fluorescein-labelled glycerol-phosphate dehydrogenase with aldolase indicated that both fructose-1,6-bisphosphate and fructose-2,6-biphosphate enhanced the affinity of aldolase to glycerol-phosphate dehydrogenase. The results of the kinetic and binding experiments suggest that binding of the C-6 phosphate group of fructose-1,6-bisphosphate to aldolase complexed with dehydrogenase is sterically impeded while saturation of the C-6 phosphate group site increases the affinity of aldolase for dehydrogenase. The possible molecular mechanism of the fructose-1,6-bisphosphate modulated interaction is discussed.

Identification of tyrosine as a functional residue in the active site of Escherichia coli dUTPase.

dUTP nucleotidohydrolase (dUTPase, EC 3.6.1.23) from E. coli contains a total of six tyrosine residues per trimer. About half of them were found to be susceptible to acetylation with N-acetylimidazole or to nitration with tetranitromethane with concomitant loss of activity. Deacetylation with N-hydroxylamine leads to full reactivation. Inhibitory products of dUTP hydrolysis, i.e., dUMP and inorganic pyrophosphate together with the cofactor Mg2+ protect significantly against inactivation and chemical modification. In the Cu(2+)-dUTPase complex, charge transfer from Cu2+ to the tyrosinate anion was perturbed by the presence of the substrate dUTP. These results, together with the occurrence of one tyrosine residue in a strictly conserved sequence motif suggest the critical importance of this residue for the function of the enzyme.

A new potent calmodulin antagonist with arylalkylamine structure: crystallographic, spectroscopic and functional studies.

An arylalkylamine-type calmodulin antagonist, N-(3, 3-diphenylpropyl)-N'-[1-R-(3, 4-bis-butoxyphenyl)ethyl]-propylene-diamine (AAA) is presented and its complexes with calmodulin are characterized in solution and in the crystal. Near-UV circular dichroism spectra show that AAA binds to calmodulin with 2:1 stoichiometry in a Ca(2+)-dependent manner. The crystal structure with 2:1 stoichiometry is determined to 2.64 A resolution. The binding of AAA causes domain closure of calmodulin similar to that obtained with trifluoperazine. Solution and crystal data indicate that each of the two AAA molecules anchors in the hydrophobic pockets of calmodulin, overlapping with two trifluoperazine sites, i.e. at a hydrophobic pocket and an interdomain site. The two AAA molecules also interact with each other by hydrophobic forces. A competition enzymatic assay has revealed that AAA inhibits calmodulin-activated phosphodiesterase activity at two orders of magnitude lower concentration than trifluoperazine. The apparent dissociation constant of AAA to calmodulin is 18 nM, which is commensurable with that of target peptides. On the basis of the crystal structure, we propose that the high-affinity binding is mainly due to a favorable entropy term, as the AAA molecule makes multiple contacts in its complex with calmodulin.

Specific characteristics of phosphofructokinase-microtubule interaction.

Muscle phosphofructokinase interacts with microtubule-associated protein-free microtubules resulting in a reduction of the overall activity of the enzyme [Lehotzky et al. (1993) J. Biol. Chem. 268, 10888-10894] and periodical cross-linking of the tubules [Lehotzky et al. (1994) Biochem. Biophys. Res. Commun. 204, 585-591]. Microtubule polymers of 'tail-free' tubulin obtained by removal of the carboxy-termini with limited subtilisin digestion retain the binding domains for phosphofructokinase that cross-bridges microtubule 'bodies'. Microtubule-associated proteins bound on tubulin 'tails' do not perturb the kinase binding. These data suggest that the tubulin carboxy-terminal domain is not involved in microtubule-phosphofructokinase interactions and phosphofructokinase and microtubule-associated proteins have distinct binding domains on microtubules. Of different isoforms of phosphofructokinase, occurring mainly in brain and tumor cells, the muscle isoform exhibits selective adsorption behaviour on microtubules. Phosphofructokinase M and C isoforms with different associative and allosteric properties may represent an auxiliary pathway to modulate energy production via glycolysis.

The complete triphosphate moiety of non-hydrolyzable substrate analogues is required for a conformational shift of the flexible C-terminus in E. coli dUTP pyrophosphatase.

The molecular mechanism of substrate analogue interaction with Escherichia coli dUTPase was investigated, using the non-hydrolyzable 2'-deoxyuridine 5'-(alpha,beta-imido)triphosphate (alpha,beta-imido-dUTP). Binding of this analogue induces a difference in the far UV circular dichroism (CD) spectrum arguing for a significant change in protein conformation. The spectral shift is strictly Mg2+-dependent, does not appear with dUDP instead of alpha,beta-imido-dUTP and is not elicited if the flexible C-terminal arm is deleted from the protein by limited tryptic digestion. Involvement of the C-terminal arm in alpha,beta-imido-dUTP binding is consistent with the finding that this analogue protects against tryptic hydrolysis at Arg-141. Near UV CD of ligand-enzyme complexes reveals a characteristic difference in the microenvironments of enzyme-bound dUDP and alpha,beta-imido-dUTP, a difference not observable in C-terminally truncated dUTPase. The results suggest that (i) closing of the active site during the catalytic cycle, through the movement of the C-terminal arm, requires the presence of the complete triphosphate moiety of the substrate in complex with Mg2+, and (ii) after catalytic cleavage the active site pops open to facilitate product release.

The interaction of a new anti-tumour drug, KAR-2 with calmodulin.

1. KAR-2 (3"-(beta-chloroethyl)-2",4"-dioxo-3,5" -spiro-oxazolidino-4-deacetoxy-vinblastine) is a semisynthetic bis-indol derivative, with high anti-microtubular and anti-tumour activities but with low toxicity. KAR-2, in contrast to other biologically active bis-indols (e.g. vinblastine) did not show anti-calmodulin activity in vitro (enzyme kinetic, fluorescence anisotropy and immunological tests). 2. Direct binding studies (fluorescence resonance energy transfer, circular dichroism) provided evidence for the binding of KAR-2 to calmodulin. The binding affinity of KAR-2 to calmodulin (dissociation constant was about 5 microM) in the presence of Ca2+ was comparable to that of vinblastine. 3. KAR-2 was able to interact with apo-calmodulin as well; in the absence of Ca2+ the binding was of cooperative nature. 4. The effect of drugs on Ca2+ homeostasis in human neutrophil cells was investigated by means of a specific fluorescent probe. Trifluoperazine extensively inhibited the elevation of intracellular Ca2+ level, vinblastine did not appreciably affect it, KAR-2 stimulated the Ca2+ influx and after a transient enhancement the Ca2+ concentration reached a new steady-state level. 5. Comparison of the data obtained with KAR-2 and bis-indols used in chemotherapy suggests that the lack of anti-calmodulin potency resides on the spiro-oxazolidino portion of KAR-2. This character of KAR-2 manifested itself in various systems and might result in its low in vivo toxicity, established in an anti-tumour test.

Interaction of a new bis-indol derivative, KAR-2 with tubulin and its antimitotic activity.

1. KAR-2 (3"-(beta-chloroethyl)-2",4"-dioxo-3,5"-spiro-oxazolidino- 4-deacetoxy-vinblastine), is a bis-indol derivative; catharantine is coupled with the vindoline moiety which contains a substituted oxazolidino group. Our binding studies showed that KAR-2 exhibited high affinity for bovine purified brain tubulin (Kd-3 microM) and it inhibited microtubule assembly at a concentration of 10 nM. 2. Anti-microtubular activity of KAR-2 was highly dependent on the ultrastructure of microtubules: while the single tubules were sensitive, the tubules cross-linked by phosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11) exhibited significant resistance against KAR-2. 3. The cytoplasmic microtubules of Chinese hamster ovary mammalian and Sf9 insect cells were damaged by 1 microgram ml-1 KAR-2, as observed by indirect immunofluorescence and transmission electron microscopy. Scanning electron microscopy revealed intensive surface blebbing on both types of cells in the presence of KAR-2. 4. KAR-2 was effective in the mouse leukaemia P338 test in vivo without significant toxicity. Studies on a primary cerebro-cortical culture of rat brain and differentiated PC12 cells indicated that the toxicity of KAR-2 was significantly lower than that of vinblastine. The additional property of KAR-2 that distinguishes it from bis-indol derivatives is the lack of anti-calmodulin activity.

Anti-calmodulin potency of indol alkaloids in in vitro systems.

We have demonstrated that bis-indol Vinca alkaloids of anti-mitotic activities (vinblastine, vincristine, and navelbine) bind to calmodulin in a Ca(2+)-dependent manner. We designed direct binding tests (fluorescence energy transfer and circular dichroism measurements) to quantify the interactions of bis-indol derivatives with calmodulin. The dissociation constants of calmodulin-navelbine and calmodulin-vinblastine complexes with 1:1 stoichiometry are 0.5 microM and 3 microM, respectively. These values indicate that the binding affinities of these Vinca alkaloids to calmodulin and tubulin are comparable. Immunological, enzyme kinetic and fluorescence anisotropy measurements showed that bis-indol alkaloids inhibit the interactions of calmodulin with target proteins. The results of indirect enzyme-linked immunosorbent assay showed that bis-indol alkaloids effectively antagonize with anti-calmodulin antibody for calmodulin binding (IC50 = 90 microM, 400 microM, and 430 microM for navelbine, vincristine and vinblastine, respectively). According to the fluorescence anisotropy and enzyme kinetic measurements, vinblastine, vincristine and vinblastine, similarly to trifluoperazine, the classic calmodulin antagonist, compete with target enzyme [phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11)] for an inhibitory effect either on immunocomplex formation or on calmodulin-enzyme interaction. Navelbine appeared in our tests as the most potent drug in inhibiting the association of calmodulin to target proteins in comparison to other bis-indol derivatives. Since navelbine and vinblastine possess identical vindoline moiety, although they differ in the catharantine part, the difference in anti-calmodulin potencies is suggested to reside predominantly on this portion of the molecules. These findings might establish the pharmacological importance of these activities in the specificity and toxicity of the drugs.

Mechanistic studies of dUTPases.

The essential enzyme dUTPase is responsible for preventive DNA repair via exclusion of uracil. Lack or inhibition of the enzyme induces thymine-less cell death in cells performing active DNA synthesis, serving therefore as an important chemotherapeutic target. In the present work, employing differential circular dichroism spectroscopy, we show that D. mel. dUTPase, a recently described eukaryotic model, has a similar affinity of binding towards alpha,beta-imino-dUTP as compared to the prokaryotic E. coli enzyme. However, in contrast to the prokaryotic dUTPase, the nucleotide exerts significant protection against tryptic digestion at a specific tryptic site 20 A far from the active site in the fly enzyme. This result indicates that binding of the nucleotide in the active site induces an allosteric conformational change within the central threefold channel of the homotrimer exclusively in the eukaryotic enzyme. Nucleotide binding induced allosterism in the D. mel. dUTPase, but not in the E. coli enzyme, might be associated with the altered hydropathy of subunit interfaces in these two proteins.

Malaria pigment crystals as magnetic micro-rotors: key for high-sensitivity diagnosis.

The need to develop new methods for the high-sensitivity diagnosis of malaria has initiated a global activity in medical and interdisciplinary sciences. Most of the diverse variety of emerging techniques are based on research-grade instruments, sophisticated reagent-based assays or rely on expertise. Here, we suggest an alternative optical methodology with an easy-to-use and cost-effective instrumentation based on unique properties of malaria pigment reported previously and determined quantitatively in the present study. Malaria pigment, also called hemozoin, is an insoluble microcrystalline form of heme. These crystallites show remarkable magnetic and optical anisotropy distinctly from any other components of blood. As a consequence, they can simultaneously act as magnetically driven micro-rotors and spinning polarizers in suspensions. These properties can gain importance not only in malaria diagnosis and therapies, where hemozoin is considered as drug target or immune modulator, but also in the magnetic manipulation of cells and tissues on the microscopic scale.

Flexible glycine rich motif of Escherichia coli deoxyuridine triphosphate nucleotidohydrolase is important for functional but not for structural integrity of the enzyme.

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), a ubiquitous enzyme of DNA metabolism, has been implicated as a novel target of anticancer and antiviral drug design. This task is most efficiently accomplished by X-ray crystallography of the relevant protein-inhibitor complexes. However, the topic of the present investigation, a glycine-rich strictly conserved structural motif of dUTPases, could not be located in the crystal structure of the Escherichia coli enzyme, probably due to its increased flexibility. The present work shows that removal of a C-terminal 11-residue fragment, including this motif, by limited trypsinolysis strongly impairs catalytic activity. Kinetic analysis of the intact and digested variants showed that kcat decreases 40-fold, while KM increases less than twofold upon digestion. The tryptic site was identified by mass spectrometry, amino acid analysis and N-terminal sequencing. The shortened enzyme variant retains the secondary, tertiary, and quaternary (trimeric) structure of the intact species as suggested by UV absorption, fluorescence and circular dichroism spectroscopy, and analytical gel filtration. Moreover, binding affinity of the shortened variant toward the substrate analogue MgdUDP is identical to the one displayed by the intact enzyme. I conclude that the glycine-rich motif is functionally relevant for E. coli dUTPase. It may play a role in enzymatic catalysis by contributing to the formation of the catalytically potent enzyme-substrate complex.

Altered subunit communication in subfamilies of trimeric dUTPases.

The enzyme dUTPase is essential in preventing uracil incorporation into DNA. Design of antagonists against this novel chemotherapeutic target requires identification of species-specific differences in the structure and mechanism of the enzyme. This task is now approached via comparisons of available crystallographic structures of dUTPases from Homo sapiens, Escherichia coli, and retroviruses. The eukaryotic protein uniquely displays polar and charged amino acid residues participating in threefold intersubunit interactions. In bacterial and retroviral dUTPases, threefold interactions are mainly hydrophobic. The residues responsible for this contrast are mapped in multiple sequence alignment to positions differently and characteristically conserved in distinct evolutionary branches. The general feature of this contrast is further strengthened by a second eukaryotic model structure constructed using comparative modeling. The dUTPase cDNA from Drosophila melanogaster was identified, sequenced, and the model structure of the encoded polypeptide displayed a polar hydrogen-bonding network of threefold interactions, identically to the human structure. Results allow clear distinction between two subfamilies of trimeric dUTPases where altered subunit communication may account for a functional difference in the catalytic cycle.

Elasticity of the human red cell membrane skeleton. Effects of temperature and denaturants.

The molecular basis for the elasticity of the human erythrocyte membrane was explored. Skeletons were released from ghosts in Triton X-100 and their dimensions followed by dark-field microscopy and packed volume. The rest size of skeletons was assumed to reflect the balance point between expansion (deformation) driven by electrostatic repulsions among the excess of fixed negative charges on the proteins and contraction (recovery) driven by their elasticity. The size of skeletons decreased with increasing temperature. This finding suggests that entropy drives elasticity. The requisite entropy change could be associated with either the configurational freedom of flexible protein chains or with the solvation of side chains exposed during protein dissociation (hydrophobic effects). To distinguish between these two alternatives, we tested the impact of two weak denaturants, 10% ethanol and 20 nM lithium 3,5-diiodosalicylate. Both agents reversibly promoted the expansion of skeletons, presumably by reducing their elasticity. Since the conformation of random coils and globular proteins should not be significantly altered by these mild treatments, this finding strongly suggests a role for weak interdomain and/or interprotein associations. We conclude that the elasticity of the red cell membrane skeleton may not derive from the configurational entropy of flexible coils. Rather, the elastic energy may arise from reversible dissociations of weak but specific intramolecular and/or intermolecular contacts, presumably within deformed spectrin filaments.

Alternative binding of two sequential glycolytic enzymes to microtubules. Molecular studies in the phosphofructokinase/aldolase/microtubule system.

Simultaneous binding of two sequential glycolytic enzymes, phosphofructokinase and aldolase, to a microtubular network was investigated. The binding of the phosphofructokinase to microtubules and its bundling activity has been previously characterized (Lehotzky, A., Telegdi, M., Liliom, K., and Ovádi, J. (1993) J. Biol. Chem. 268, 10888-10894). Aldolase binding to microtubules at near physiological ionic strength is weak (Kd = 20 microM) as compared with that of the kinase (Kd = 1 microM). The interactions of both enzymes with microtubules are modulated by their common intermediate, fructose-1,6-bisphosphate. Pelleting and electron microscopic measurements have revealed that the aldolase binding interferes with that of phosphofructokinase, although they have distinct binding domains on microtubules. The underlying molecular mechanism responsible for this finding is that in the solution phase aldolase and phosphofructokinase form a bienzyme complex that does not bind to the microtubule. The bienzyme complex formation does not influence the catalytic activity of aldolase, however, it inhibits the dissociation-induced inactivation of the kinase by stabilizing a catalytically active molecular form. The present data suggest the first experimental evidence that two sequential glycolytic enzymes do not associate simultaneously to microtubules, but their complexation in solution provides kinetic advantage for glycolysis.

Triosephosphate isomerase deficiency: predictions and facts.

Deficiencies in around 20 enzymes, associated with widely different degrees of severity and complexity, have been identified for human erythrocytes. The fact that glycolysis is crucial for erythrocyte function is reflected by the large number of inherited glycolytic enzymopathies. Triosephosphate isomerase (TPI) deficiency, a rare autosomal disease, is usually associated with nonspherocytic hemolytic anemia, progressive neurologic dysfunction, and death in childhood. The two affected Hungarian brothers studied by us have less than 3% TPI activity and enormously (30-50-fold) increased dihydroxyacetone phosphate (DHAP) concentration in their erythrocytes. The well-established concept of the metabolic control theory was used to test the contribution of TPI and some related enzymes to the control of a relevant segment of the glycolytic pathway in normal and deficient cells. Deviation indices, DEJ = (delta J/delta E) E(r)/J(r), which give a good estimation of flux control coefficients using a single large change in enzyme activity, were determined from the fluxes in the absence and presence of exogeneous enzymes. We found that PFK and aldolase are the enzymes that predominantly control the flux, however, the quantitative values depend extensively on the pH: DEJ values are 0.85 and 0.14 at pH 8.0 and 0.33 and 0.67 at pH 7.2 for aldolase and PFK, respectively. Neither the flux rates nor the capacities of the enzymes seem to be significantly different in normal and TPI deficient cells. There is a discrepancy between DHAP levels and TPI activities in the deficient cells. In contrast to the experimental data the theoretical calculations predict elevation in DHAP level at lower than 0.1% of the normal value of TPI activity. Several possibilities suggested fail to explain this discrepancy. Specific associations of glycolytic enzymes to band-3 membrane proteins with their concomitant inactivation have been demonstrated. We propose that the microcompartmentation of TPI that could further decrease the reduced isomerase activity of the deficient cells, is responsible for the high DHAP level.

Pyruvate kinase as a microtubule destabilizing factor in vitro.

Endogenous control of microtubule dynamism is essential in many cell types. Numerous microtubule-adhering proteins stabilize the polymer status, while very few protein factors are described with opposite effects. The brain- and muscle-specific M1 isoform of the enzyme pyruvate kinase is investigated here in this respect. Three pieces of evidence indicate antimicrotubular effects of this protein. (1) Pyruvate kinase inhibits taxol-induced tubulin polymerization into microtubules as revealed by turbidimetry. (2) Pelleting experiments show that pyruvate kinase partially disassembles taxol-stabilized microtubules into less sedimentable oligomers leading to the appearance of tubulin in the supernatant fractions. (3) Electron microscopy reveals the kinase-induced formation of great amounts of thread-like tubulin oligomers which tend to accumulate in a light/less sedimentable fraction. Immunoelectron micrographs using labeled antibody against pyruvate kinase provide evidence for the binding of pyruvate kinase to the thread-like oligomeric forms. The present data allow the assumption that pyruvate kinase may display multiple regulatory functions as a glycolytic control enzyme and as a modulator of microtubule dynamism.

Simultaneous binding of drugs with different chemical structures to Ca2+-calmodulin: crystallographic and spectroscopic studies.

The modulatory action of Ca2+-calmodulin on multiple targets is inhibited by trifluoperazine, which competes with target proteins for calmodulin binding. The structure of calmodulin crystallized with two trifluoperazine molecules is determined by X-ray crystallography at 2.74 A resolution. The X-ray data together with the characteristic and distinct signals obtained by circular dichroism in solution allowed us to identify the binding domains as well as the order of the binding of two trifluoperazine molecules to calmodulin. Accordingly, the binding of trifluperazine to the C-terminal hydrophobic pocket is followed by the interaction of the second drug molecule with an interdomain site. Recently, we demonstrated that the two bisindole derivatives, vinblastine and KAR-2 [3"-(beta-chloroethyl)-2",4"-dioxo-3, 5"-spirooxazolidino-4-deacetoxyvinblastine], interact with calmodulin with comparable affinity; however, they display different functional effects [Orosz et al. (1997) British J. Pharmacol. 121, 955-962]. The structural basis responsible for these effects were investigated by circular dichroism and fluorescence spectroscopy. The data provide evidence that calmodulin can simultaneously accommodate trifluoperazine and KAR-2 as well as vinblastine and KAR-2, but not trifluoperazine and vinblastine. The combination of the binding and structural data suggests that distinct binding sites exist on calmodulin for vinblastine and KAR-2 which correspond, at least partly, to that of trifluoperazine at the C-terminal hydrophobic pocket and at an interdomain site, respectively. This structural arrangement can explain why these drugs display different anticalmodulin activities. Calmodulin complexed with melittin is also able to bind two trifluoperazine molecules, the binding of which appears to be cooperative. Results obtained with intact and proteolytically cleaved calmodulin reveal that the central linker region of the protein is indispensable for simultanous interactions with two molecules of either identical or different ligands.

Specific derivatization of the active site tyrosine in dUTPase perturbs ligand binding to the active site.

Selective modification of one (of three) tyrosine residue per enzyme monomer leads to inactivation of dUTPase of the retrovirus equine infectious anemia virus (EIAV). The substrate dUMP and the cofactor Mg2+ protect against inactivation and modification, in agreement with the study on E. coli dUTPase (Vertessy et al. (1994) Biochim. Biophys. Acta 1205, 146-150). Amino acid analyses of nitrated dUTPases confirmed Tyr-selectivity of modification. The nitrated residue in E. coli dUTPase was identified as the evolutionary highly conserved Tyr-93. The modifiable residue is shown to be the only Tyr exposed in both E. coli and EIAV dUTPases. As a consequence of Tyr-93 derivatization, the Mg2+-dependent interaction between the substrate-analogue dUDP and E. coli dUTPase becomes impaired as shown by circular dichroism spectroscopy, here presented as a tool for monitoring ligand binding to the active site.

Crystallization and preliminary diffraction analysis of Ca(2+)-calmodulin-drug and apocalmodulin-drug complexes.

Ca(2+)-calmodulin is crystallized with two new and potent drugs: a bisindol derivative (KAR-2, 3"-(beta-chloroethyl)-2",4"-dioxo-3,5"- spiro-oxazolidino-4-deacetoxy-vinblastine) with antitumor activity and an arylalkylamine fendiline analogue (N-(3,3-diphenylpropyl)-N'-[1-(3,4- di-n-butoxy-phenyl)-ethyl]-1,3-diaminopropane) with anticalmodulin activity. The crystals diffract beyond 2.8 A and differ in unit cell parameters from each other as well as from crystals of Ca(2+)-calmodulin or Ca(2+)-calmodulin-ligand complexes, as reported thus far. Attempts to crystallize Ca(2+)-free calmodulin without drugs failed, in consonance with earlier results; however, single Ca(2+)-free calmodulin crystals diffracting-beyond 2.5 A resolution were grown in the presence of KAR-2. Results indicate that binding of the two drugs to apocalmodulin or Ca(2+)-calmodulin may induce unique novel protein conformers, targets of further detailed X-ray studies.